Nuclear actin dynamics and functions at a glance.

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

Successful delivery of viviparous lantern shark from an artificial uterus and the self-production of lantern shark luciferin.

Our recent success in the long-term maintenance of lantern shark embryos in artificial uterine systems has provided a novel option for the medical treatment of premature embryos for captive viviparous elasmobranchs. The remaining issue with this system is that the embryos cannot survive the abrupt change in the chemical environment from artificial uterine fluid (AUF) to seawater during delivery. To overcome this issue, the present study developed a new protocol for seawater adaptation, which is characterized by a long-term and stepwise shift from AUF to seawater prior to delivery. This protocol was employed successfully, and the specimen survived for more than seven months after delivery, the longest captive record of the species. During the experiment, we unexpectedly detected bioluminescence of the embryonic lantern shark in the artificial uterus. This observation indicates that lantern sharks can produce luciferin, a substance for bioluminescence. This contradicts the recent hypothesis that lantern sharks lack the ability to produce luciferin and use luciferin obtained from food sources.

Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

Atlantoaxial Instability after Treatment of Atlantoaxial Rotatory Fixation: A Case Report.

Introduction: We encountered a case of atlantoaxial subluxation (AAS) after treatment of atlantoaxial rotatory fixation (AARF). Reports of developing AAS after AARF are extremely rare. Case Report: An 8-year-old male who feels neck pain was diagnosed with AARF type II according to the Fielding classification. Computed tomography (CT) showed that the atlas was rotated 32° to the right relative to the axis. Neck collar, Glisson traction, and reduction under anesthesia were performed. Five months after the onset of AARF, the patient was diagnosed with AAS due to dilatation of atlantodental interval (ADI) and underwent posterior cervical fusion. Conclusion: AARF treatments, such as long-term Glisson traction and reduction under general anesthesia, which exert a stress on the cervical spine, may damage the alar ligaments, apical ligaments, lower longitudinal band, and Gruber's ligament. Transverse ligament damage can also occur during the treatment of AARF, especially if AARF is refractory or requires long-term treatment. In addition, knowledge of the pathophysiology of atlantoaxial instability after AARF treatment is important.

A Rare Case of Cervical Myelopathy Caused by an Anomaly of the Axis: A Case Report and a Review of Current Comprehensive Literature.

Introduction: Cervical myelopathy due to anomalies of the posterior elements of the axis is rare, and limited cases have been reported. This study reports such a unique case of congenital anomaly of the laminae of the axis, which is treated by partial removal of the anomalous bony structure surgically, and provides a comprehensive review of the present literature on this pathological condition. Case Presentation: A 65-year-old man presented with a 3-year history of numbness in both arms with a sensory change in the right arm. Six months before admission, he noticed weakness in his right upper extremities and stiffness of the right lower extremity. Magnetic resonance imaging (MRI) showed a bilaterally compressed spinal cord with an anomalous bony structure at the C2-3 level. Computed tomography (CT) showed an invaginated abnormal bony structure at the C2-3 level and an abnormal lateral mass on the right side at the C2-3 level. The patient underwent posterior decompression surgery using the conventional open approach. One year after surgery, his myelopathy partially ameliorated, with his cervical radiograph showing no signs of the secondary instability and MRI showing sufficient decompression of the spinal cord. The age of onset, symptoms, and surgical treatment in our case was similar to those in the 14 previously reported cases; however, the morphology of the anomaly had variations. Conclusion: This is a report of a rare anomaly in the laminae of the axis. MRI and reconstructed CT scans were useful for the treatment of this case. Partial surgical removal was an appropriate treatment for this patient, resulting in a satisfactory outcome.

Actin nucleoskeleton in embryonic development and cellular differentiation.

Dynamic assembly and disassembly of actin proteins play a key role in the cytoskeleton, but the cellular functions of actin are not only restricted to the cytoplasmic compartment. Recent studies have shown that actin spatiotemporally changes its polymerized state in the nucleus as well and such dynamic nature of actin is relevant to key nuclear events including gene expression, DNA damage response and chromatin organization. In this review, we highlight emerging roles of actin in the nuclear compartment especially in the context of embryonic development and cellular differentiation. We first explain how the actin nucleoskeleton can be formed and function in cells. Notably, nuclear actin dynamics are greatly altered when cell fates change, such as after fertilization and T cell differentiation. We discuss how the dynamic actin nucleoskeleton contributes to accomplishing developmental programs.

Single-cell profiling of transcriptomic changes during in vitro maturation of human oocytes.

Purpose: In vitro maturation (IVM) of human oocytes offers an invaluable opportunity for infertility treatment. However, in vitro matured oocytes often show lower developmental abilities than their in vivo counterparts, and molecular mechanisms underlying successful maturation remain unclear. In this study, we investigated gene expression profiles of in vitro matured oocytes at the single-cell level to gain mechanistic insight into IVM of human oocytes. Methods: Human oocytes were retrieved by follicular puncture and in vitro matured. In total, 19 oocytes from 11 patients were collected and subjected to single-cell RNA-seq analyses. Results: Global gene expression profiles were similar among oocytes at the same maturation stage, while a small number of oocytes showed distinct transcriptomes from those at the corresponding maturation stage. Differential gene expression analysis identified hundreds of transcripts that dynamically altered their expression during IVM, and we revealed molecular pathways and upstream regulators that may govern oocyte maturation. Furthermore, oocytes that were delayed in their maturation showed distinct transcriptomes. Finally, we identified genes whose transcripts were enriched in each stage of oocyte maturation. Conclusions: Our work uncovers transcriptomic changes during human oocyte IVM and the differential gene expression profile of each oocyte.

Nuclear transfer system for the direct induction of embryonic transcripts from intra- and cross-species nuclei using mouse 4-cell embryos.

Reprogramming of somatic nuclei toward the embryonic state has been studied using nuclear transfer (NT) to an oocyte at the metaphase II (MII) stage. In this NT, a somatic nucleus transplanted into an MII oocyte of the same species undergoes DNA replication and cell division before activating embryonic genes. Here, we describe a direct NT protocol using 4-cell stage mouse embryos that enables reprogramming of intra- and cross-species nuclei to express embryonic genes without requiring DNA replication and cell division. For complete details on the use and execution of this protocol, please refer to Tomikawa et al. (2021).

Compensatory Pelvic Retro-Rotation Associated with a Decreased Quality of Life in Patients with Normal Sagittal Balance.

STUDY DESIGN: Cross-sectional observational study. PURPOSE: To examine whether pelvic rotation as a compensatory mechanism for sagittal imbalance is related to quality of life (QOL). OVERVIEW OF LITERATURE: Poor sagittal alignment is associated with compensatory pelvic retroversion and decreased QOL. Whether the compensatory pelvic tilt (PT) influences QOL is unclear. METHODS: Overall, 134 subjects aged ≥20 years with lower back pain were included (104 females; mean age, 70±9.8 years). Sagittal vertical alignment (SVA) and PT were analyzed radiographically. Patients were stratified into three groups based on SVA values: good alignment (group G), intermediate alignment (group I), and poor sagittal alignment (group P). Patients in group I were further categorized into two groups: low PT and high PT. The Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ) was used for clinical assessment, and the scores were compared between groups. RESULTS: As SVA increased, PT and lumbar lordosis (LL) increased and decreased, respectively. PT and LL differed significantly between groups G and P (p<0.01 for each comparison). Within group I, there was no significant difference in SVA between the high PT and low PT groups, suggesting that the high PT group had acquired a compensated sagittal balance. Importantly, all domains in the JOABPEQ (except for lower back pain) were significantly lower in the high PT group than in the low PT group (p<0.05 for every comparison). CONCLUSIONS: This study showed that focusing solely on SVA as a single indicator can cause important losses in QOL to be overlooked in patients with lumbar disorders. Although pelvic retroversion can compensate for sagittal balance, it is associated with a significant decrease in QOL. To improve the assessment of patients with lumbar disorders, PT should be considered besides SVA.

Structural alteration of the nucleus for the reprogramming of gene expression.

The regulation of gene expression is a critical process for establishing and maintaining cellular identity. Gene expression is controlled through a chromatin-based mechanism in the nucleus of eukaryotic cells. Recent studies suggest that chromatin accessibility and the higher-order structure of chromatin affect transcriptional outcome. This is especially evident when cells change their fate during development and nuclear reprogramming. Furthermore, non-chromosomal contents of the cell nucleus, namely nucleoskeleton proteins, can also affect chromatin and nuclear structures, resulting in transcriptional alterations. Here, we review our current mechanistic understanding about how chromatin and nuclear structures impact transcription in the course of embryonic development, cellular differentiation and nuclear reprogramming, and also discuss unresolved questions that remain to be addressed in the field.

Cell division- and DNA replication-free reprogramming of somatic nuclei for embryonic transcription.

Nuclear transfer systems represent the efficient means to reprogram a cell and in theory provide a basis for investigating the development of endangered species. However, conventional nuclear transfer using oocytes of laboratory animals does not allow reprogramming of cross-species nuclei owing to defects in cell divisions and activation of embryonic genes. Here, we show that somatic nuclei transferred into mouse four-cell embryos arrested at the G2/M phase undergo reprogramming toward the embryonic state. Remarkably, genome-wide transcriptional reprogramming is induced within a day, and ZFP281 is important for this replication-free reprogramming. This system further enables transcriptional reprogramming of cells from Oryx dammah, now extinct in the wild. Thus, our findings indicate that arrested mouse embryos are competent to induce intra- and cross-species reprogramming. The direct induction of embryonic transcripts from diverse genomes paves a unique approach for identifying mechanisms of transcriptional reprogramming and genome activation from a diverse range of species.

Ectopic gas in the fibular graft after anterior cervical corpectomy and fusion.

BACKGROUND: Ectopic gas in the graft is occasionally encountered upon follow-up computed tomography (CT) after anterior cervical corpectomy and fusion (ACCF). However, most cases lack inflammatory responses and manifestations of infection. Although the clinical significance of ectopic gas in the graft has not yet been established, to the best of our knowledge, no previous studies have described ectopic gas in the graft after ACCF. This study evaluated ectopic gas in the fibular graft upon follow-up CT after ACCF. METHODS: We reviewed 112 patients who underwent ACCF and follow-up CT, with a minimum follow-up period of 3 years. CT images were retrospectively reviewed to confirm the presence of ectopic gas in the graft and bone fusion. Bone fusion was defined as follows: mobility less than 2 mm between spinous processes on the flection-extension radiograph or a bone bridge on CT images. RESULTS: Of the 112 patients, 30 (27%) patients had ectopic gas in the fibular grafts. Among them, ectopic gas was initially observed 3 months after surgery (early onset) in 23 (77%) patients and 6 months after surgery (late-onset) in the remaining seven (23%) patients. Upon the latest follow-up CT, ectopic gas more frequently remained in late-onset (4/7, 57%) rather than in early-onset (3/23, 13%) cases (p = 0.033). Bone fusion was not observed when CT images exhibited ectopic gas in the graft, whereas ectopic gas was not observed when CT images exhibited bone fusion. CONCLUSION: Ectopic gas in the fibular graft was observed at both early and late-onset after ACCF; late-onset gas remained significantly. The remaining gas was strongly associated with pseudoarthrosis; therefore, pseudoarthrosis should be considered when ectopic gas in the graft is observed on CT images.

Volume of the whale shark and their mechanism of vertical feeding.

The present study provides a noninvasive method to estimate the body volume of sharks (Elasmobranchii, Selachii) using a computational geometric model. This method allows the volume of sharks to be estimated from lateral and ventral photographs assuming an elliptical body cross-sectional geometry. A comparison of the estimated and actual body volumes of several shark species showed that the estimation error was < 0.5%. The accuracy of the model decreased if photographs that were inclined to the orthogonal plane were used, although this error was on average < 2.3% if the inclination angle was 10° or less. Applying this model to captive whale sharks (Rhincodon typus) that were 8.0 and 8.8 m in total length revealed that their body volumes were 3.5 and 4.5 m3, respectively. These estimates allowed for the quantitative evaluation of our hypothesis, that the whale shark uses suctioned air for buoyancy control during vertical feeding-a behavior unique to this species among elasmobranchs. The volume estimates of the captive whale sharks, together with the density estimates from their liver proportions, revealed that the air occupying a part of oro-pharyngeal and branchial cavities can help the whale sharks to keep their body floating. This hypothesis may explain how the whale shark sometimes stays at the water surface without fin motion during vertical feeding, even though their body density is greater than that of seawater.

Symmetrically dimethylated histone H3R2 promotes global transcription during minor zygotic genome activation in mouse pronuclei.

Paternal genome reprogramming, such as protamine-histone exchange and global DNA demethylation, is crucial for the development of fertilised embryos. Previously, our study showed that one of histone arginine methylation, asymmetrically dimethylated histone H3R17 (H3R17me2a), is necessary for epigenetic reprogramming in the mouse paternal genome. However, roles of histone arginine methylation in reprogramming after fertilisation are still poorly understood. Here, we report that H3R2me2s promotes global transcription at the 1-cell stage, referred to as minor zygotic genome activation (ZGA). The inhibition of H3R2me2s by expressing a histone H3.3 mutant H3.3R2A prevented embryonic development from the 2-cell to 4-cell stages and significantly reduced global RNA synthesis and RNA polymerase II (Pol II) activity. Consistent with this result, the expression levels of MuERV-L as minor ZGA transcripts were decreased by forced expression of H3.3R2A. Furthermore, treatment with an inhibitor and co-injection of siRNA to PRMT5 and PRMT7 also resulted in the attenuation of transcriptional activities with reduction of H3R2me2s in the pronuclei of zygotes. Interestingly, impairment of H3K4 methylation by expression of H3.3K4M resulted in a decrease of H3R2me2s in male pronuclei. Our findings suggest that H3R2me2s together with H3K4 methylation is involved in global transcription during minor ZGA in mice.

Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

Nucleoskeleton proteins for nuclear dynamics.

The eukaryotic nucleus shows organized structures of chromosomes, transcriptional components and their associated proteins. It has been believed that such a dense nuclear environment prevents the formation of a cytoskeleton-like network of protein filaments. However, accumulating evidence suggests that the cell nucleus also possesses structural filamentous components to support nuclear organization and compartments, which are referred to as nucleoskeleton proteins. Nucleoskeleton proteins including lamins and actin influence nuclear dynamics including transcriptional regulation, chromatin organization and DNA damage responses. Furthermore, these nucleoskeleton proteins play a pivotal role in cellular differentiation and animal development. In this commentary, we discuss how nucleoskeleton-based regulatory mechanisms orchestrate nuclear dynamics.

Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer.

Actin in the nucleus, referred to as nuclear actin, is involved in a variety of nuclear events. Nuclear actin is present as a globular (G-actin) and filamentous form (F-actin), and dynamic assembly/disassembly of nuclear actin profoundly affects nuclear functions. However, it is still challenging to observe endogenous nuclear F-actin. Here, we present a condition to visualize endogenous nuclear F-actin of mouse zygotes using different fixation methods. Zygotes fixed with paraformaldehyde and treated with fluorescently conjugated phalloidin show both short and long actin filaments in their pronuclei. Short nuclear actin filaments are characteristic of phalloidin staining, rather than the consequence of severing actin filaments by the fixation process, since long nuclear actin filaments probed with the nuclear actin chromobody are not disassembled into short filaments after fixation with paraformaldehyde. Furthermore, we find that nuclear actin assembly is impaired after somatic cell nuclear transfer (SCNT), suggesting abnormal nucleoskeleton structures in SCNT embryos. Taken together, our presented method for visualizing nuclear F-actin with phalloidin can be used to observe the states of nuclear actin assembly, and revealed improper reprogramming of actin nucleoskeleton structures in cloned mouse embryos.

Parascolopsis akatamae, a new species of dwarf monocle bream (Perciformes: Nemipteridae) from the Indo-West Pacific, with redescription of closely related species P. eriomma.

A new species of dwarf monocle bream, Parascolopsis akatamae n. sp., is described from the Indo-West Pacific. The new species is distinguished from all other species of Parascolopsis in having 16-19 gill rakers on the first arch, length of forked part of caudal fin 5.8-6.5 times in standard length, eye diameter 1.3-1.8 times in length of the longest dorsal-fin spine, and a pale yellow stripe present from lower edge of the eye to posterior edge of the preopercle. Parascolopsis eriomma (Jordan Richardson, 1909) is morphologically very similar to the new species and the two have been confused with each other for a long time. Therefore, we redescribe P. eriomma based on the holotype and newly collected specimens. In addition, we found that patterns of biofluorescence emission for both species are clearly different. This suggests that their biofluorescence patterns may function in distinguishing each other.

Impairment of nuclear F-actin formation and its relevance to cellular phenotypes in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a mutation of lamin A, which contributes to nuclear architecture and the spatial organization of chromatin in the nucleus. The expression of a lamin A mutant, named progerin, leads to functional and structural disruption of nuclear organization. Since progerin lacks a part of the actin-binding site of lamin A, we hypothesized that nuclear actin dynamics and function are altered in HGPS cells. Nuclear F-actin is required for the organization of nuclear shape, transcriptional regulation, DNA damage repair, and activation of Wnt/ß-catenin signaling. Here we show that the expression of progerin decreases nuclear F-actin and impairs F-actin-regulated transcription. When nuclear F-actin levels are increased by overexpression of nuclear-targeted actin or by using jasplakinolide, a compound that stabilizes F-actin, the irregularity of nuclear shape and defects in gene expression can be reversed. These observations provide evidence for a novel relationship between nuclear actin and the etiology of HGPS.

Zygotic Nuclear F-Actin Safeguards Embryonic Development.

After fertilization, sperm and oocyte nuclei are rapidly remodeled to form swollen pronuclei (PN) in mammalian zygotes, and the proper formation and function of PN are key to producing totipotent zygotes. However, how mature PN are formed has been unclear. We find that filamentous actin (F-actin) assembles in the PN of mouse zygotes and is required for fully functional PN. The perturbation of nuclear actin dynamics in zygotes results in the misregulation of genes related to genome integrity and abnormal development of mouse embryos. We show that nuclear F-actin ensures DNA damage repair, thus preventing the activation of a zygotic checkpoint. Furthermore, optogenetic control of cofilin nuclear localization reveals the dynamically regulated F-actin nucleoskeleton in zygotes, and its timely disassembly is needed for developmental progression. Nuclear F-actin is a hallmark of totipotent zygotic PN, and the temporal regulation of its polymerized state is necessary for normal embryonic development.