Avian species are essential resources for human society, with their preservation and utilization heavily dependent on primordial germ cells (PGCs). However, efficient methods for isolating live PGCs from embryos remain elusive in avian species beyond chickens, and even in chickens, existing techniques have shown limited efficiency. In this study, we present a rapid, simple, and cost-effective method for labeling and sorting circulating-stage PGCs across various avian species, including Carinatae and Ratitae, using Lycopersicon Esculentum (Tomato) lectin (LEL). Notably, this method demonstrates high sorting efficiency by identifying a wide range of PGC subtypes while preserving the proliferative and migratory potential of chicken PGCs. This approach is anticipated to significantly contribute to the conservation, research, and agricultural industries related to avian species globally.

Chicken embryos have many advantages in the study of amniote embryonic development. In particular, culture techniques developed for early-stage embryos have promoted the advancement of modern developmental studies using chicken embryos. However, the standard technique involves placing chicken embryos in the ventral-upward (ventral-up) orientation, limiting manipulation of the epiblast at the dorsal surface, which is the primary source of ectodermal and mesodermal tissues. To circumvent this limitation, we developed chicken embryo cultures in the dorsal-up orientation and exploited this technique to address diverse issues. In this article, we first review the history of chicken embryo culture techniques to evaluate the advantages and limitations of the current standard technique. Then, the dorsal-up technique is discussed. These technological discussions are followed by three different examples of experimental analyses using dorsal-up cultures to illustrate their advantages: (1) EdU labeling of epiblast cells to assess potential variation in the cell proliferation rate; (2) migration behaviors of N1 enhancer-active epiblast cells revealed by tracking cells with focal fluorescent dye labeling in dorsal-up embryo culture; and (3) neural crest development of mouse neural stem cells in chicken embryos.

Both development and regeneration depend on signaling centers, which are sources of locally secreted tissue-patterning molecules. As many signaling centers are decommissioned before the end of embryogenesis, a fundamental question is how signaling centers can be re-induced later in life to promote regeneration after injury. Here, we use the axolotl salamander model (Ambystoma mexicanum) to address how the floor plate is assembled for spinal cord regeneration. The floor plate is an archetypal vertebrate signaling center that secretes Shh ligand and patterns neural progenitor cells during embryogenesis. Unlike mammals, axolotls continue to express floor plate genes (including Shh) and downstream dorsal-ventral patterning genes in their spinal cord throughout life, including at steady state. The parsimonious hypothesis that Shh+ cells give rise to functional floor plate cells for regeneration had not been tested. Using HCR in situ hybridization and mathematical modeling, we first quantified the behaviors of dorsal-ventral spinal cord domains, identifying significant increases in gene expression level and floor plate size during regeneration. Next, we established a transgenic axolotl to specifically label and fate map Shh+ cells in vivo. We found that labeled Shh+ cells gave rise to regeneration floor plate, and not to other neural progenitor domains, after tail amputation. Thus, despite changes in domain size and downstream patterning gene expression, Shh+ cells retain their floor plate identity during regeneration, acting as a stable cellular source for this regeneration signaling center in the axolotl spinal cord.

Mitochondria are unique organelles that have their own genome (mtDNA) and perform various pivotal functions within a cell. Recently, evidence has highlighted the role of mitochondria in the process of stem cell differentiation, including differentiation of neural stem cells (NSCs). Here we studied the importance of mtDNA function in the early differentiation process of NSCs in two cell culture models: the CGR8-NS cell line that was derived from embryonic stem cells by a lineage selection technique, and primary NSCs that were isolated from embryonic day 14 mouse fetal forebrain. We detected a dramatic increase in mtDNA content upon NSC differentiation to adapt their mtDNA levels to their differentiated state, which was not accompanied by changes in mitochondrial transcription factor A expression. As chemical mtDNA depletion by ethidium bromide failed to generate living ρ° cell lines from both NSC types, we used inhibition of mtDNA polymerase-γ by 2'-3'-dideoxycytidine to reduce mtDNA replication and subsequently cellular mtDNA content. Inhibition of mtDNA replication upon NSC differentiation reduced neurogenesis but not gliogenesis. The mtDNA depletion did not change energy production/consumption or cellular reactive oxygen species (ROS) content in the NSC model used. In conclusion, mtDNA replication is essential for neurogenesis but not gliogenesis in fetal NSCs through as yet unknown mechanisms, which, however, are largely independent of energy/ROS metabolism.

Cardiovascular disease is the leading cause of mortality worldwide. Myocardial injury resulting from ischemia can be fatal because of the limited regenerative capacity of adult myocardium. Mammalian cardiomyocytes rapidly lose their proliferative capacities, with only a small fraction of adult myocardium remaining proliferative, which is insufficient to support post-injury recovery. Recent investigations have revealed that this decline in myocardial proliferative capacity is closely linked to perinatal metabolic shifts. Predominantly glycolytic fetal myocardial metabolism transitions towards mitochondrial fatty acid oxidation postnatally, which not only enables efficient production of ATP but also causes a dramatic reduction in cardiomyocyte proliferative capacity. Extensive research has elucidated the mechanisms behind this metabolic shift, as well as methods to modulate these metabolic pathways. Some of these methods have been successfully applied to enhance metabolic reprogramming and myocardial regeneration. This review discusses recently acquired insights into the interplay between metabolism and myocardial proliferation, emphasizing postnatal metabolic transitions.

Experimental evolution of microbial cells provides valuable information on evolutionary dynamics, such as mutations that contribute to fitness gain under given selection pressures. Although experimental evolution is a promising tool in evolutionary biology and bioengineering, long-term culture experiments under multiple environmental conditions often impose an excessive workload on researchers. Therefore, the development of automated systems significantly contributes to the advancement of experimental evolutionary research. This review presents several specialized devices designed for experimental evolution studies, such as an automated system for high-throughput culture experiments, a culture device that generate a temperature gradient, and an automated ultraviolet (UV) irradiation culture device. The ongoing development of such specialized devices is poised to continually expand new frontiers in experimental evolution research.

The 57th Annual Meeting of the Japanese Society for Developmental Biologists was held at Miyako Messe and ROHM Theater Kyoto from June 19 to 22, 2024. After the COVID-19 pandemic, this was the first meeting where all restrictions were removed. This year's theme was "Breaking Through Boundaries." It was set to be enjoyed by everyone across academic fields and nationalities. About 600 people from 16 countries participated in this meeting. Four workshops, 117 posters, 16 oral sessions (80 titles), 7 symposia, 2 plenary lectures, and various other formats ware included. I would like to share some of the lively discussions and wonderful sessions that I attended.

The development of new technologies opens new avenues in the research field. Gene knockout is a key method for analyzing gene function in mice. Currently, conditional gene knockout strategies are employed to examine temporal and spatial gene function. However, phenotypes are sometimes not observed because of the time required for depletion due to the long half-life of the target proteins. Protein knockdown using an improved auxin-inducible degron system, AID2, overcomes such difficulties owing to rapid and efficient target depletion. We observed depletion of AID-tagged proteins within a few to several hours by a simple intraperitoneal injection of the auxin analog, 5-Ph-IAA, which is much shorter than the time required for target depletion using conditional gene knockout. Importantly, the loss of protein is reversible, making protein knockdown useful to measure the effects of transient loss of protein function. Here, we also established several mouse lines useful for AID2-medicated protein knockdown, which include knock-in mouse lines in the ROSA26 locus; one expresses TIR1(F74G), and the other is the reporter expressing AID-mCherry. We also established a germ-cell-specific TIR1 line and confirmed the protein knockdown specificity. In addition, we introduced an AID tag to an endogenous protein, DCP2 via the CAS9-mediated gene editing method. We confirmed that the protein was effectively eliminated by TIR1(F74G), which resulted in the similar phenotype observed in knockout mouse within 20 h.

In recent decades, developmental biologists have come to view vascular development as a series of progressive transitions. Mesoderm differentiates into endothelial cells; arteries, veins and lymphatic endothelial cells are specified from early endothelial cells; and vascular networks diversify and invade developing tissues and organs. Our understanding of this elaborate developmental process has benefitted from detailed studies using the zebrafish as a model system. Here, we review a number of key developmental transitions that occur in zebrafish during the formation of the blood and lymphatic vessel networks.

The brain in the genus Homo expanded rapidly during evolution, accelerated by a reciprocated interaction between neural, cognitive, and ecological niches (triadic niche construction, or TNC). This biologically costly expansion incubated latent cognitive capabilities that, with a quick and inexpensive rewiring of brain areas in a second phase of TNC, provided the basis for Homo sapiens specific abilities. The neural demands for perception of the human body in interaction with tools and the environment required highly integrated sensorimotor domains, inducing the parietal lobe expansion seen in humans. These newly expanded brain areas allowed connecting the sensations felt in the body to the actions in the world through the cognitive function of "projection". In this opinion article, we suggest that as a relationship of equivalence between body parts, tools and their external effects was established, mental mechanisms of self-objectification might have emerged as described previously, grounding notions of spatial organization, idealized objects, and their transformations, as well as socio-emotional states in the sensing agent through a self-in-the-world map. Therefore, human intelligence and its features such as symbolic thought, language, mentalizing, and complex technical and social behaviors could have stemmed from the explicit awareness of the causal relationship between the self and intentional modifications to the environment.

Parkinson's disease is a neurological disorder characterized by reduced motility, depression and dementia. Guamanian parkinsonism dementia with amyotrophic sclerosis is a local case of Parkinson's disease reported in the Western Pacific Islands of Guam and Rota as well as in the Kii Peninsula of Japan. A previous genetic study has suggested that Guamanian parkinsonism is attributable to a variant of the TRPM7 gene, which encodes for melastatin-related transient receptor potential (TRP) ion channels. But the link between parkinsonism and the TRPM7 gene remains elusive. Previous studies have addressed that trpm7-deficient zebrafish embryos showed defects in pigmentation and touch-evoked motor response. In this study, we identified a new viable allele of trpm7 mutant causing an I756N amino acid substitution in the first transmembrane domain. Behavioral analyses revealed that trpm7 mutants showed compromised motility with their movement distance shorter than wild-type larvae. The velocity of the movement was significantly reduced in trpm7 mutants than in wild-type larvae. Along with a previous finding of reduced dopaminergic neurons in zebrafish trpm7 mutants, reduced motility of trpm7 mutants can suggest another similarity between trpm7 phenotypes and Parkinson's disease symptoms.

One of the major functions of the semaphorin signaling system is the regulation of cell shape. In the nematode Caenorhabditis elegans, membrane-bound semaphorins SMP-1/2 (SMPs) regulate the morphology of epidermal cells via their receptor plexin, PLX-1. In the larval male tail of the SMP-PLX-1 signaling mutants, the border between two epidermal cells, R1.p and R2.p, is displaced anteriorly, resulting in the anterior displacement of the anterior-most ray, ray 1, in the adult male. To elucidate how the intercellular signaling mediated by SMPs regulates the position of the intercellular border, we performed mosaic gene expression analyses by using infrared laser-evoked gene operator (IR-LEGO). We show that PLX-1 expressed in R1.p and SMP-1 expressed in R2.p are required for the proper positioning of ray 1. The result suggests that SMP signaling promotes extension, rather than retraction, of R1.p. This is in contrast to a previous finding that SMPs mediate inhibition of cell extension of vulval precursor cells, another group of epidermal cells of C. elegans, indicating the context dependence of cell shape control via the semaphorin signaling system.

Microglia colonize the brain starting on embryonic day (E) 9.5 in mice, and their population increases with development. We have previously demonstrated that some microglia are derived from intraventricular macrophages, which frequently infiltrate the pallium at E12.5. To address how the infiltration of intraventricular macrophages is spatiotemporally regulated, histological analyses detecting how these cells associate with the surrounding cells at the site of infiltration into the pallial surface are essential. Using two-photon microscopy-based in vivo imaging, we demonstrated that most intraventricular macrophages adhere to the ventricular surface. This is a useful tool for imaging intraventricular macrophages maintaining their original position, but this method cannot be used for observing deeper brain regions. Meanwhile, we found that conventional cryosection-based and naked pallial slice-based observation resulted in unexpected detachment from the ventricular surface of intraventricular macrophages and their mislocation, suggesting that previous histological analyses might have failed to determine their physiological number and location in the ventricular space. To address this, we sought to establish a methodological preparation that enables us to delineate the structure and cellular interactions when intraventricular macrophages infiltrate the pallium. Here, we report that brain slices pretreated with agarose-embedding maintained adequate density and proper positioning of intraventricular macrophages on the ventricular surface. This method also enabled us to perform the immunostaining. We believe that this is helpful for conducting histological analyses to elucidate the mechanisms underlying intraventricular macrophage infiltration into the pallium and their cellular properties, leading to further understanding of the process of microglial colonization into the developing brain.

During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non-neural ectoderm and that it increased in an actomyosin activity-dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large-scale morphogenetic movements.

Oviposition is induced upon mating in most insects. Spawning is a physiological process that is fundamental for the reproduction of Scylla paramamosain. However, the molecular mechanisms underlying the spawning process in this species are poorly understood. Herein, comprehensive ovary transcriptomic analysis was conducted at the germinal vesicle breakdown stage (GVBD), spawning stage, 0.5 h post-spawning stage, and 24 h post-spawning stage of S. paramamosain for gene discovery. A total of 67,230 unigenes were generated, and 27,975 (41.61%) unigenes were annotated. Meanwhile, the differentially expressed genes (DEGs) between the different groups were identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was subsequently conducted. These results suggested that octopamine (OA) and tyramine (TA) could induce oviposition, while dopamine (DA) and serotonin (5-hydroxytryptamine [5-HT]) inhibit oviposition. The 20-hydroxyecdysone (20E) and methyl farnesoate (MF) signal pathways might be positively associated with oviposition. Furthermore, numerous transcripts that encode neuropeptides and their G-protein-coupled receptors (GPCRs), such as CNMamide, RYamide, ecdysis-triggering hormone (ETH), GPA2/GPB5 receptor, and Moody receptor, appear to be differentially expressed during the spawning process. Eleven unigenes were selected for qRT-PCR and the pattern was found to be consistent with the transcriptome expression pattern. Our work is the first spawning-related investigation of S. paramamosain focusing on the ovary at the whole transcriptome level. These findings assist in improving our understanding of spawning regulation in S. paramamosain and provide information for oviposition studies in other crustaceans.

The northern house gecko Hemidactylus flaviviridis exhibits appendage-specific responses to injuries. The autotomized tail regenerates, whereas the severed limb fails to regrow. Many site-specific cellular processes influence tail regeneration. Herein, we analyzed the epithelial-mesenchymal transition contrast in the lizard's amputated appendages (tail and limb). Morphological observations in the healing frame indicated the formation of regeneration blastema in the tail and scar formation in limb. Histology of the tail showed that epithelial cells closer to mesenchyme appeared less columnar and loosely packed, with little intercellular matrix. Whereas in the limb, the columnar epithelial cells remained tightly packed. Collagen deposition was seen in the limb at the intersection of wound epithelium and mesenchyme, favoring scarring by blocking the epithelial-mesenchymal transition. Markers for epithelial-mesenchymal transition were assessed at transcript and protein levels. The regenerating tail showed upregulation of N-cadherin, vimentin, and PCNA, favoring epithelial-mesenchymal transition, cell migration, and proliferation, respectively. In contrast, the scarring limb showed persistently elevated levels of E-cadherin and EpCAM, indicating retention of epithelial characteristics. An attempt was made to screen the resident epithelial stem cell population in both appendages to check their potential role in the epithelial-mesenchymal transition (EMT), hence the differential wound healing. Upregulation in transcript and protein levels of Nanog and Sox2 was observed in the regenerating tail. Fluorescence-activated cell sorting (FACS) provided supporting evidence that the epithelial stem cell population in tail remained significantly higher than in limb. Thus, this study focuses on the mechanistic role of the epithelial-mesenchymal transition in wound healing, highlighting the molecular details of regeneration and scarring events.

The update of the draft genome assembly of sea urchin, Hemicentrotus pulcherrimus, which is widely studied in East Asia as a model organism of early development, was performed using Oxford nanopore long-read sequencing. The updated assembly provided ~600-Mb genome sequences divided into 2,163 contigs with N50 = 516 kb. BUSCO completeness score and transcriptome model mapping ratio (TMMR) of the present assembly were obtained as 96.5% and 77.8%, respectively. These results were more continuous with higher resolution than those by the previous version of H. pulcherrimus draft genome, HpulGenome_v1, where the number of scaffolds = 16,251 with a total of ~100 Mb, N50 = 143 kb, BUSCO completeness score = 86.1%, and TMMR = 55.4%. The obtained genome contained 36,055 gene models that were consistent with those in other echinoderms. Additionally, two tandem repeat sequences of early histone gene locus containing 47 copies and 34 copies of all histone genes, and 185 of the homologous sequences of the interspecifically conserved region of the Ars insulator, ArsInsC, were obtained. These results provide further advance for genome-wide research of development, gene regulation, and intranuclear structural dynamics of multicellular organisms using H. pulcherrimus.

Vision is formed by the transmission of light stimuli to the brain through axons extending from photoreceptor cells. Damage to these axons leads to loss of vision. Despite research on neural circuit regeneration through transplantation, achieving precise axon projection remains challenging. To achieve optic nerve regeneration by transplantation, we employed the Drosophila visual system. We previously established a transplantation method for Drosophila utilizing photoreceptor precursor cells extracted from the eye disc. However, little axonal elongation of transplanted cells into the brain, the lamina, was observed. We verified axonal elongation to the lamina by modifying the selection process for transplanted cells. Moreover, we focused on N-cadherin (Ncad), a cell adhesion factor, and Twinstar (Tsr), which has been shown to promote actin reorganization and induce axon elongation in damaged nerves. Overexpression of Ncad and tsr promoted axon elongation to the lamina, along with presynaptic structure formation in the elongating axons. Furthermore, overexpression of Neurexin-1 (Nrx-1), encoding a protein identified as a synaptic organizer, was found to not only promote presynapse formation but also enhance axon elongation. By introducing Ncad, tsr, and Nrx-1, we not only successfully achieved axonal projection of transplanted cells to the brain beyond the retina, but also confirmed the projection of transplanted cells into a deeper ganglion, the medulla. The present study offers valuable insights to realize regeneration through transplantation in a more complex nervous system.

In this study, we comprehensively searched for fish-specific genes in gnathostomes that contribute to development of the fin, a fish-specific trait. Many previous reports suggested that animal group-specific genes are often important for group-specific traits. Clarifying the roles of fish-specific genes in fin development of gnathostomes, for example, can help elucidate the mechanisms underlying the formation of this trait. We first identified 91 fish-specific genes in gnathostomes by comparing the gene repertoire in 16 fish and 35 tetrapod species. RNA-seq analysis narrowed down the 91 candidates to 33 genes that were expressed in the developing pectoral fin. We analyzed the functions of approximately half of the candidate genes by loss-of-function analysis in zebrafish. We found that some of the fish-specific and fin development-related genes, including fgf24 and and1/and2, play roles in fin development. In particular, the newly identified fish-specific gene qkia is expressed in the developing fin muscle and contributes to muscle morphogenesis in the pectoral fin as well as body trunk. These results indicate that the strategy of identifying animal group-specific genes is functional and useful. The methods applied here could be used in future studies to identify trait-associated genes in other animal groups.

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.