Evidence of a novel silencing effect on transgenes in the Arabidopsis thaliana sperm cell.

We encountered unexpected transgene silencing in Arabidopsis thaliana sperm cells; transgenes encoding proteins with no specific intracellular localization (cytoplasmic proteins) were silenced transcriptionally or posttranscriptionally. The mRNA of cytoplasmic protein transgenes tagged with a fluorescent protein gene was significantly reduced, resulting in undetectable fluorescent protein signals in the sperm cell. Silencing of the cytoplasmic protein transgenes in the sperm cell did not affect the expression of either its endogenous homologous genes or cotransformed transgenes encoding a protein with targeted intracellular localization. This transgene silencing in the sperm cell persisted in mutants of the major gene silencing machinery including DNA methylation. The incomprehensible, yet real, transgene silencing phenotypes occurring in the sperm cell could mislead the interpretation of experimental results in plant reproduction, and this Commentary calls attention to that risk and highlights details of this novel cytoplasmic protein transgene silencing.

Prominent caudal shift of the lumbar plexus roots in spines with 18 thoracolumbar vertebrae.

PURPOSE: It remains unclear whether concomitant changes in the thoracolumbar (TL) vertebrae and lumbar plexus roots seen in experimental embryology are present in humans with different vertebral formulas, particularly in humans with 18 TL vertebrae. We thus investigated the human lumbar plexus root changes occurring in spines with an additional TL vertebra (18TL). METHODS: The lumbosacral plexus was macroscopically dissected in TL anomaly cases found in 161 computed tomography examinations. TL anomalies were distinguished as simple abnormalities in total TL count and abnormal TL trade-offs, i.e., exchanges between the last thoracic and first lumbar vertebrae, and were analyzed separately. RESULTS: One additional TL vertebra (7C_18TL_5S) was observed in 4/159 cases (2.5%), excluding cases with cervical and sacral abnormalities. Different from the unclear shifts of nerve roots in cases with 16TL and 17TL trade-offs, the 18TL trade-off tended to involve a caudal shift at the cranial limit, without event change at the caudal limit. In addition, only one nerve segment shift was reconfirmed with a change in two vertebral segments from 16 to 18 TL vertebrae. CONCLUSIONS: We revealed that concomitant changes in the lumbar plexus roots and vertebrae in humans with 18TL vertebrae may become more pronounced than those in humans with 16 or 17TL vertebrae, by approaching the typical mammalian TL formula (19TL). This study showed that the TL formula can be used to estimate changes in the lumbar plexus roots, which may assist in the planning of nerve-sparing spinal and pelvic surgery.

Cellular dynamics of coenocytic endosperm development in Arabidopsis thaliana.

After double fertilization, the endosperm in the seeds of many flowering plants undergoes repeated mitotic nuclear divisions without cytokinesis, resulting in a large coenocytic endosperm that then cellularizes. Growth during the coenocytic phase is strongly associated with the final seed size; however, a detailed description of the cellular dynamics controlling the unique coenocytic development in flowering plants has remained elusive. By integrating confocal microscopy live-cell imaging and genetics, we have characterized the entire development of the coenocytic endosperm of Arabidopsis thaliana including nuclear divisions, their timing intervals, nuclear movement and cytoskeleton dynamics. Around each nucleus, microtubules organize into aster-shaped structures that drive actin filament (F-actin) organization. Microtubules promote nuclear movement after division, while F-actin restricts it. F-actin is also involved in controlling the size of both the coenocytic endosperm and the mature seed. The characterization of cytoskeleton dynamics in real time throughout the entire coenocyte endosperm period provides foundational knowledge of plant coenocytic development, insights into the coordination of F-actin and microtubules in nuclear dynamics, and new opportunities to increase seed size and our food security.

F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus (FA) facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study, we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in FA formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the FA and loss of polar AtLURE1.2 distribution toward the FA. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male-female communication in the synergid cell.

Live-cell imaging reveals the cellular dynamics in seed development.

Seed development in flowering plants is highly complex and governed by three genetically distinct tissues: the fertilization products, the diploid embryo and triploid endosperm, as well as the seed coat that has maternal origin. There are diverse cellular dynamics such as nuclear movement in gamete cells for fertilization, cell polarity establishment for embryo development, and multinuclear endosperm formation. These tissues also coordinate and synchronize the developmental timing for proper seed formation through cell-to-cell communications. Live-cell imaging using advanced microscopy techniques enables us to decipher the dynamics of these events. Especially, the establishment of a less-invasive semi-in vivo live-cell imaging approach has allowed us to perform time-lapse analyses for long period observation of Arabidopsis thaliana intact seed development dynamics. Here we highlight the recent trends of live-cell imaging for seed development and discuss where we are heading.

Effect of assimilate competition during early seed development on the pod and seed growth traits in soybean.

Although the seed remains small in size during the initial stage of seed development (the lag phase), several studies indicate that environment and assimilate supply level manipulations during the lag phase affect the final seed size. However, the manipulations were not only at the lag phase, making it difficult to understand the specific role of the lag phase in final seed size determination. It also remained unclear whether environmental cues are sensed by plants and regulate seed development or if it is simply the assimilate supply level, changed by the environment, that affects the subsequent seed development. We investigated soybean (Glycine max L. Merr.) seed phenotypes grown in a greenhouse using different source-sink manipulations (shading and removal of flowers and pods) during the lag phase. We show that assimilate supply is the key factor controlling flower and pod abortion and that the assimilate supply during the lag phase affects the subsequent potential seed growth rate during the seed filling phase. In response to low assimilate supply, plants adjust flower/pod abortion and lag phase duration to supply the minimum assimilate per pod/seed. Our results provide insight into the mechanisms whereby the lag phase is crucial for seed development and final seed size potential, essential parameters that determine yield.

Integrated anatomical practice combining cadaver dissection and matched cadaver CT data processing and analysis.

PURPOSE: With the increasing significance of diagnostic imaging in clinical practice, long-term anatomical education and training is required to ensure that students can reliably distinguish anatomical structures and interpret images. To improve students' motivation and prospects for learning imaging anatomy, we developed an integrated anatomical practice program combining cadaveric dissection with cadaver CT data processing and analysis during undergraduate students' dissection courses. METHODS: Workstations imported with post-mortem CT data of dissected cadavers and various forms of clinical CT/MRI data were set in the dissection room. Medical students had free access to the imaging data during cadaver dissection, and they were challenged to process and analyze the data for submission of voluntary imaging reports on their topics of interest. Finally, we surveyed the integrated anatomical education of 481 medical students. RESULTS: The positive response rate to the integrated anatomical practice was 74.9%, and 79.4% of the students answered that this form of practice offered a suitable introduction to anatomical imaging. The usefulness of this approach in understanding the 2- to 3D arrangement of the human body and enhancing interest in anatomy was also confirmed. The submission rate of voluntary imaging reports also increased annually and is currently 97.4%. CONCLUSION: Our integrated anatomical practice only allowed students to actively browse CT images and facilitated imaging processing and analysis of their region of interest. This practice may improve students' long-term ability to analyze images and deepen their understanding. A competitive imaging contest may help improve students' motivation when they begin learning imaging anatomy.

Comparative transcriptomic analysis reveals conserved programmes underpinning organogenesis and reproduction in land plants.

The appearance of plant organs mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes and the development of novel regulatory programmes. However, no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we generated gene expression atlases for various organs and gametes of ten plant species comprising bryophytes, vascular plants, gymnosperms and flowering plants. A comparative analysis of the atlases identified hundreds of organ- and gamete-specific orthogroups and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, our results suggest that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, which indicates that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function.

Insights into the molecular evolution of fertilization mechanism in land plants.

KEY MESSAGE: Comparative genetics and genomics among green plants, including algae, provide deep insights into the evolution of land plant sexual reproduction. Land plants have evolved successive changes during their conquest of the land and innovations in sexual reproduction have played a major role in their terrestrialization. Recent years have seen many revealing dissections of the molecular mechanisms of sexual reproduction and much new genomics data from the land plant lineage, including early diverging land plants, as well as algae. This new knowledge is being integrated to further understand how sexual reproduction in land plants evolved, identifying highly conserved factors and pathways, but also molecular changes that underpinned the emergence of new modes of sexual reproduction. Here, we review recent advances in the knowledge of land plant sexual reproduction from an evolutionary perspective and also revisit the evolution of angiosperm double fertilization.

Formins control dynamics of F-actin in the central cell of Arabidopsis thaliana.

In the female gamete of flowering plants, sperm nuclear migration is controlled by a constant inward movement of actin filaments (F-actin) for successful fertilization. This dynamic F-actin movement is ARP2/3-independent, raising the question of how actin nucleation and polymerization is controlled in the female gamete. Using confocal microscopy live-cell imaging in combination with a pharmacological approach, we assessed the involvement of another group of actin nucleators, formins, in F-actin inward movement in the central cell of Arabidopsis thaliana. We identify that the inhibition of the formin function, by formin inhibitor SMIFH2, significantly reduced the dynamic inward movement of F-actin in the central cell, indicating that formins play a major role in actin nucleation required for F-actin inward movement in the central cell.

First in situ 3D visualization of the human cardiac conduction system and its transformation associated with heart contour and inclination.

Current advanced imaging modalities with applied tracing and processing techniques provide excellent visualization of almost all human internal structures in situ; however, the actual 3D internal arrangement of the human cardiac conduction system (CCS) is still unknown. This study is the first to document the successful 3D visualization of the CCS from the sinus node to the bundle branches within the human body, based on our specialized physical micro-dissection and its CT imaging. The 3D CCS transformation by cardiac inclination changes from the standing to the lying position is also provided. Both actual dissection and its CT image-based simulation identified that when the cardiac inclination changed from standing to lying, the sinus node shifted from the dorso-superior to the right outer position and the atrioventricular conduction axis changed from a vertical to a leftward horizontal position. In situ localization of the human CCS provides accurate anatomical localization with morphometric data, and it indicates the useful correlation between heart inclination and CCS rotation axes for predicting the variable and invisible human CCS in the living body. Advances in future imaging modalities and methodology are essential for further accurate in situ 3D CCS visualization.

Postmortem evaluation of neuromuscular damages more extensive than the surgical intervention area after iliac crest bone graft.

Several complications may occur following iliac bone grafting, one of the common sites for autologous bone harvesting. Of these, it is difficult to localize the damage in neurological complications due to the presence of several nerves in a similar distribution area with variations among individuals. To minimize these complications, conventional clinical anatomical studies using normal human cadavers have estimated the theoretical neurological damage area corresponding to the surgical intervention area. We report a case of neuromuscular damage in a 93-year-old woman who had an iliac crest defect after a bone graft, based on the virtual and physical dissections with histological confirmations.In this study, the patient was confirmed to have severe neuromuscular complications with major complications including a hernia protruding through the iliac defect. One of the two ilioinguinal nerves was extracted with the hernia sac through the iliac defect, and its distal part was completely damaged. The iliohypogastric nerve, which was far from the defect foramen, also showed remarkable fibrosis and demyelination, affected by the degeneration of the transversus abdominis muscles.The present anatomical findings show that the area of eventual neuromuscular damage should be estimated to larger than the conventionally predicted area of direct nerve damage, which is usually concomitant with the surgical intervention area.

Cellular dynamics of double fertilization and early embryogenesis in flowering plants.

Flowering plants (angiosperms) perform a unique double fertilization in which two sperm cells fuse with two female gamete cells in the embryo sac to develop a seed. Furthermore, during land plant evolution, the mode of sexual reproduction has been modified dramatically from motile sperm in the early-diverging land plants, such as mosses and ferns as well as some gymnosperms (Ginkgo and cycads) to nonmotile sperm that are delivered to female gametes by the pollen tube in flowering plants. Recent studies have revealed the cellular dynamics and molecular mechanisms for the complex series of double fertilization processes and elucidated differences and similarities between animals and plants. Here, together with a brief comparison with animals, we review the current understanding of flowering plant zygote dynamics, covering from gamete nuclear migration, karyogamy, and polyspermy block, to zygotic genome activation as well as asymmetrical division of the zygote. Further analyses of the detailed molecular and cellular mechanisms of flowering plant fertilization should shed light on the evolution of the unique sexual reproduction of flowering plants.

ARP2/3-independent WAVE/SCAR pathway and class XI myosin control sperm nuclear migration in flowering plants.

After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration in Arabidopsis thaliana (Arabidopsis) and Nicotiana tabacum (tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in the Arabidopsis central cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.

Plasmogamic Paternal Contributions to Early Zygotic Development in Flowering Plants.

Flowering plant zygotes possess complete developmental potency, and the mixture of male and female genetic and cytosolic materials in the zygote is a trigger to initiate embryo development. Plasmogamy, the fusion of the gamete cytoplasms, facilitates the cellular dynamics of the zygote. In the last decade, mutant analyses, live cell imaging-based observations, and direct observations of fertilized egg cells by in vitro fusion of isolated gametes have accelerated our understanding of the post-plasmogamic events in flowering plants including cell wall formation, gamete nuclear migration and fusion, and zygotic cell elongation and asymmetric division. Especially, it has become more evident that paternal parent-of-origin effects, via sperm cytoplasm contents, not only control canonical early zygotic development, but also activate a biparental signaling pathway critical for cell fate determination after the first cell division. Here, we summarize the plasmogamic paternal contributions via the entry of sperm contents during/after fertilization in flowering plants.

Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin.

Epigenetic marks are reprogrammed in the gametes to reset genomic potential in the next generation. In mammals, paternal chromatin is extensively reprogrammed through the global erasure of DNA methylation and the exchange of histones with protamines1,2. Precisely how the paternal epigenome is reprogrammed in flowering plants has remained unclear since DNA is not demethylated and histones are retained in sperm3,4. Here, we describe a multi-layered mechanism by which H3K27me3 is globally lost from histone-based sperm chromatin in Arabidopsis. This mechanism involves the silencing of H3K27me3 writers, activity of H3K27me3 erasers and deposition of a sperm-specific histone, H3.10 (ref. 5), which we show is immune to lysine 27 methylation. The loss of H3K27me3 facilitates the transcription of genes essential for spermatogenesis and pre-configures sperm with a chromatin state that forecasts gene expression in the next generation. Thus, plants have evolved a specific mechanism to simultaneously differentiate male gametes and reprogram the paternal epigenome.

The phenotypic morphology of human lumbar plexus roots associated with changes in the thoracolumbar vertebral count and trade-off.

This study investigated the developmental basis for the human phenotypic morphology of the interaction between the vertebrae and the nerve plexus by evaluating changes in the human lumbar plexus according to various thoracolumbar formulas. The dissection found that the changes in lumbar nerve roots reported by experimental embryology studies to be concomitant with thoracolumbar trade-off, i.e., a change in vertebrae from thoracic to lumbar with no change in the overall thoracolumbar count, were not apparent in humans with the usual 17 or mutant 16 thoracolumbar vertebrae. When vertebral changes in two segments were examined by comparing spines with a reduced thoracolumbar count of 16 to those with an increased count of 18, this tended to show only a single-segment caudal shift of the lumbar plexus. We cannot provide evidence for the phylogenetic difference in the concomitant changes of lumbar nerves and vertebrae, but comparisons between experimental rodents and humans highlighted fewer and shorter lumbar vertebra and more complicated lumbar plexus in humans. Therefore, these multiple differences may contribute to a human phenotypic morphology that is not evident in the concomitant transformation of vertebrae and lumbar nerves reported in experimental rodents.

Derived muscle arrangements and their shared innervation patterns of external and internal cheek pouches in rodents.

Cheek pouches have evolved from the oral cavity in rodents and act as temporary food storage repositories. There are two types of opening, internal and external. Details about the complex cutaneous muscles controlling the pouches have still not been fully elucidated. To understand the shared and derived traits of the muscles surrounding the cheek pouch and their innervation, we carried out an evolutionary morphological study using two desert kangaroo rats (Dipodomys deserti) and three plains pocket gophers (Geomys bursarius) from each of the two families equipped with external cheek pouches, and four Syrian hamsters (Mesocricetus auratus) with internal cheek pouches. The most conspicuous derived trait of the muscles between the external and internal cheek pouches, the sphincter sacculi, surrounds almost all of the edge of the outer entrance of the pouch. It is present in both species with external pouches, but not in hamsters, which have internal pouches. Our neurological findings demonstrate that most pouch muscles are innervated by both the facial and the cervical nerves, regardless of the pouch type. In these dually innervated muscles, the ventromedial part of the muscles tends to be innervated dominantly or uniquely by the cervical nerves, which usually enter from the superficial or lateral aspect. As a trait shared with the cervical nerves innervating the propatagial muscles in aerodynamic mammals such as bats and flying squirrels, and panniculus carnosus in most mammals, our neurological evidence suggests that the cervical nerve has the potential to innervate derived cutaneous muscles in the cervicofacial region.