Effect of Temperature on the Embryogenesis of Three Geographically Distinct Populations of Meloidogyne incognita Is Driven by Intrinsic Thermal Acclimation Reaction.

Research interest in the mechanisms enabling plant-parasitic nematodes to adjust their physiological performance and cope with changing temperatures has intensified in light of global warming. Here, we show that geographically distinct populations of the root-knot nematode Meloidogyne incognita, which is prevalent in the three main pepper-growing regions in Israel-Carmel Valley (Carmel), Jordan Valley (JV), and Arava Rift (Arava)-possess persistent differences in their thermal acclimation capacity, which affect pre- and postembryonic development. The optimal temperature for embryonic growth completion was 25°C for the Carmel population; 25 and 30°C for the JV population; and 30°C for the Arava population. Cumulative hatching percentages showed variations among populations; relative to hatching at 25°C, the Carmel population experienced hatching reduction at the higher studied temperatures 30 and 33°C, while the JV and Arava populations exhibited an increase in hatching at 30 and 33°C, respectively. Juvenile survival indicates that at the lowest temperature (20°C), the Carmel population gained the highest survival rates throughout the experimental duration, while at the same duration at 33°C, the Arava population gained the highest survival rate. Infective juveniles of the Carmel population demonstrated increased penetration of tomato roots at 25°C compared to the JV and Arava populations. Inversely, at 33°C, increased penetration was observed for the Arava compared to the Carmel and JV populations. Altogether, the Arava population's performance at 33°C might incur distinct fitness costs, resulting in consistent attenuation compared to the Carmel population at 25°C. Precisely defining a population's thermal acclimation response might provide essential information for models that predict the impact of future climate change on these populations.

Transcriptomic profiling reveals histone acetylation-regulated genes involved in somatic embryogenesis in Arabidopsis thaliana.

BACKGROUND: Somatic embryogenesis (SE) exemplifies the unique developmental plasticity of plant cells. The regulatory processes, including epigenetic modifications controlling embryogenic reprogramming of cell transcriptome, have just started to be revealed. RESULTS: To identify the genes of histone acetylation-regulated expression in SE, we analyzed global transcriptomes of Arabidopsis explants undergoing embryogenic induction in response to treatment with histone deacetylase inhibitor, trichostatin A (TSA). The TSA-induced and auxin (2,4-dichlorophenoxyacetic acid; 2,4-D)-induced transcriptomes were compared. RNA-seq results revealed the similarities of the TSA- and auxin-induced transcriptomic responses that involve extensive deregulation, mostly repression, of the majority of genes. Within the differentially expressed genes (DEGs), we identified the master regulators (transcription factors - TFs) of SE, genes involved in biosynthesis, signaling, and polar transport of auxin and NITRILASE-encoding genes of the function in indole-3-acetic acid (IAA) biosynthesis. TSA-upregulated TF genes of essential functions in auxin-induced SE, included LEC1/LEC2, FUS3, AGL15, MYB118, PHB, PHV, PLTs, and WUS/WOXs. The TSA-induced transcriptome revealed also extensive upregulation of stress-related genes, including those related to stress hormone biosynthesis. In line with transcriptomic data, TSA-induced explants accumulated salicylic acid (SA) and abscisic acid (ABA), suggesting the role of histone acetylation (Hac) in regulating stress hormone-related responses during SE induction. Since mostly the adaxial side of cotyledon explant contributes to SE induction, we also identified organ polarity-related genes responding to TSA treatment, including AIL7/PLT7, RGE1, LBD18, 40, HB32, CBF1, and ULT2. Analysis of the relevant mutants supported the role of polarity-related genes in SE induction. CONCLUSION: The study results provide a step forward in deciphering the epigenetic network controlling embryogenic transition in somatic cells of plants.

Stress granule-related genes during embryogenesis of an invertebrate chordate.

Controlling global protein synthesis through the assembly of stress granules represents a strategy adopted by eukaryotic cells to face various stress conditions. TIA 1-related nucleolysin (TIAR), tristetraprolin (TTP), and Ras-GTPase-activating protein SH3-domain-binding protein (G3BP) are key components of stress granules, allowing the regulation of mRNA stability, and thus controlling not only stress responses but also cell proliferation and differentiation. In this study, we aimed at investigating the roles of tiar, ttp, and g3bp during embryogenesis of the solitary ascidian Ciona robusta under both physiological and stress conditions. We carried out CRISPR/Cas9 to evaluate the effects of gene knockout on normal embryonic development, and gene reporter assay to study the time and tissue specificity of gene transcription, together with whole-mount in situ hybridization and quantitative real time PCR. To induce acute stress conditions, we used iron and cadmium as "essential" and "non-essential" metals, respectively. Our results highlight, for the first time, the importance of tiar, ttp, and g3bp in controlling the development of mesendodermal tissue derivatives during embryogenesis of an invertebrate chordate.

Dysembryogenetic Pathogenesis of Basal Cell Carcinoma: The Evidence to Date.

The Basal Cell Carcinoma (BCC) is a sort of unique tumour due to its combined peculiar histological features and clinical behaviour, such as the constant binary involvement of the epithelium and the stroma, the virtual absence of metastases and the predilection of specific anatomical sites for both onset and spread. A potential correlation between the onset of BCC and a dysembryogenetic process has long been hypothesised. A selective investigation of PubMed-indexed publications supporting this theory retrieved 64 selected articles published between 1901 and 2024. From our analysis of the literature review, five main research domains on the dysembryogenetic pathogenesis of BCC were identified: (1) The correlation between the topographic distribution of BCC and the macroscopic embryology, (2) the correlation between BCC and the microscopic embryology, (3) the genetic BCC, (4) the correlation between BCC and the hair follicle and (5) the correlation between BCC and the molecular embryology with a specific focus on the Hedgehog signalling pathway. A large amount of data from microscopic and molecular research consistently supports the hypothesis of a dysembryogenetic pathogenesis of BCC. Such evidence is promoting advances in the clinical management of this disease, with innovative targeted molecular therapies on an immune modulating basis being developed.

The pentatricopeptide repeat protein DG1 promotes the transition to bilateral symmetry during Arabidopsis embryogenesis through GUN1-mediated plastid signals.

During Arabidopsis embryogenesis, the transition of the embryo's symmetry from radial to bilateral between the globular and heart stage is a crucial event, involving the formation of cotyledon primordia and concurrently the establishment of a shoot apical meristem (SAM). However, a coherent framework of how this transition is achieved remains to be elucidated. In this study, we investigated the function of DELAYED GREENING 1 (DG1) in Arabidopsis embryogenesis using a newly identified dg1-3 mutant. The absence of chloroplast-localized DG1 in the mutants led to embryos being arrested at the globular or heart stage, accompanied by an expansion of WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) expression. This finding pinpoints the essential role of DG1 in regulating the transition to bilateral symmetry. Furthermore, we showed that this regulation of DG1 may not depend on its role in plastid RNA editing. Nevertheless, we demonstrated that the DG1 function in establishing bilateral symmetry is genetically mediated by GENOMES UNCOUPLED 1 (GUN1), which represses the transition process in dg1-3 embryos. Collectively, our results reveal that DG1 functionally antagonizes GUN1 to promote the transition of the Arabidopsis embryo's symmetry from radial to bilateral and highlight the role of plastid signals in regulating pattern formation during plant embryogenesis.

ULI-ssDRIP-seq revealed R-loop dynamics during vertebrate early embryogenesis.

R-loop, a chromatin structure containing one RNA:DNA hybrid and one unpaired single-stranded DNA, plays multiple biological roles. However, due to technical limitations, the landscapes and potential functions of R-loops during embryogenesis remain elusive. Here, we developed a quantitative and high-resolution ultra-low input R-loop profiling method, named ULI-ssDRIP-seq, which can map global R-loops with as few as 1000 cells. By using ULI-ssDRIP-seq, we reveal the R-loop dynamics in the zebrafish from gametes to early embryos. In oocytes, the R-loop level is relatively low in most regions of the nuclear genome, except maternal-inherited rDNA and mitochondrial genome. The correlation between R-loop and CG methylation dynamics during early development is relatively weak. Furthermore, either up- or down-regulation of global R-loops by knockdown or overexpression of RNase H1 causes a delay of embryonic development with dramatic expression changes in zygotic and maternal genes. This study provides comprehensive R-loop landscapes during early vertebrate embryogenesis and demonstrates the implication of R-loops in embryonic development.

Genome-wide and functional analysis of late embryogenesis abundant (LEA) genes during dormancy and sprouting periods of kernel consumption apricots (P. armeniaca L. × P. sibirica L.).

Late embryogenesis abundant (LEA) proteins play a crucial role in protecting cells from stress, making them potential contributors to abiotic stress tolerance. This study focuses on apricot (P. armeniaca L. × P. sibirica L.), where a comprehensive genome-wide analysis identified 54 LEA genes, categorized into eight subgroups based on phylogenetic relationships. Synteny analysis revealed 14 collinear blocks containing LEA genes between P. armeniaca × P. sibirica and Arabidopsis thaliana, with an additional 9 collinear blocks identified between P. armeniaca × P. sibirica and poplar. Examination of gene structure and conserved motifs indicated that these subgroups exhibit consistent exon-intron patterns and shared motifs. The expansion and duplication of LEA genes in P. armeniaca × P. sibirica were driven by whole-genome duplication (WGD), segmental duplication, and tandem duplication events. Expression analysis, utilizing RNA-seq data and quantitative real-time RT-PCR (qRT-PCR), indicated induction of PasLEA2-20, PasLEA3-2, PasLEA6-1, Pasdehydrin-3, and Pasdehydrin-5 in flower buds during dormancy and sprouting phases. Coexpression network analysis linked LEA genes with 15 cold-resistance genes. Remarkably, during the four developmental stages of flower buds in P. armeniaca × P. sibirica - physiological dormancy, ecological dormancy, sprouting period, and germination stage - the expression patterns of all PasLEAs coexpressed with cold stress-related genes remained consistent. Protein-protein interaction networks, established using Arabidopsis orthologs, emphasized connections between PasLEA proteins and cold resistance pathways. Overexpression of certain LEA genes in yeast and Arabidopsis conferred advantages under cold stress, including increased pod length, reduced bolting time and flowering time, improved survival and seed setting rates, elevated proline accumulation, and enhanced antioxidative enzymatic activities. Furthermore, these overexpressed plants exhibited upregulation of genes related to flower development and cold resistance. The Y1H assay confirmed that PasGBF4 and PasDOF3.5 act as upstream regulatory factors by binding to the promoter region of PasLEA3-2. PasDOF2.4, PasDnaJ2, and PasAP2 were also found to bind to the promoter of Pasdehydrin-3, regulating the expression levels of downstream genes. This comprehensive study explores the evolutionary relationships among PasLEA genes, protein interactions, and functional analyses during various stages of dormancy and sprouting in P. armeniaca × P. sibirica. It offers potential targets for enhancing cold resistance and manipulating flower bud dormancy in this apricot hybrid.

The molecular biology of NF2/Merlin on tumorigenesis and development.

The neurofibromatosis type 2 (NF2) gene, known for encoding the tumor suppressor protein Merlin, is central to the study of tumorigenesis and associated cellular processes. This review comprehensively examines the multifaceted role of NF2/Merlin, detailing its structural characteristics, functional diversity, and involvement in various signaling pathways such as Wnt/ß-catenin, Hippo, TGF-ß, RTKs, mTOR, Notch, and Hedgehog. These pathways are crucial for cellular growth, proliferation, and differentiation. NF2 mutations are specifically linked to the development of schwannomas, meningiomas, and ependymomas, although the precise mechanisms of tumor formation in these specific cell types remain unclear. Additionally, the review explores Merlin's role in embryogenesis, highlighting the severe developmental defects and embryonic lethality caused by NF2 deficiency. The potential therapeutic strategies targeting these genetic aberrations are also discussed, emphasizing inhibitors of mTOR, HDAC, and VEGF as promising avenues for treatment. This synthesis of current knowledge underscores the necessity for ongoing research to elucidate the detailed mechanisms of NF2/Merlin and develop effective therapeutic strategies, ultimately aiming to improve the prognosis and quality of life for individuals with NF2 mutations.

An Introduction to Plant Cell, Tissue, and Organ Culture: Current Status and Perspectives.

Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.

Plant Micropropagation and Temporary Immersion Systems.

Temporary immersion systems (TIS) have been widely recognized as a promising technology for micropropagation of various plant species. The TIS provides a suitable environment for culture and allows intermittent contact of the explant with the culture medium at different immersion frequencies and aeration of the culture in each cycle. The frequency or immersion is one of the most critical parameters for the efficiency of these systems. The design, media volume, and container capacity substantially improve cultivation efficiency. Different TIS have been developed and successfully applied to micropropagation in various in vitro systems, such as sprout proliferation, microcuttings, and somatic embryos. TIS increases multiplication and conversion rates to plants and a better response during the ex vitro acclimatization phase. This article covers the use of different immersion systems and their applications in plant biotechnology, particularly in plant tissue culture, as well as its use in the massive propagation of plants of agroeconomic interest.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

Protocol for the Acclimatization of Coconut Vitro-Plants Under Ex Vitro Conditions.

The coconut tree is a crop widely distributed in more than 90 countries worldwide. It has a high economic value derived from the large number of products obtained from the plant, with fast-growing global markets for some of them. Unfortunately, coconut production is decreasing mainly due to the old age of the plants and devastating pests and diseases, such as phytoplasma disease lethal yellowing (LY). Massive replanting is required with phytoplasma-resistant and high-yielding selected coconut plants to keep up with the market demand for fruit. For this purpose, an efficient micropropagation technology via somatic embryogenesis has been established at CICY, yielding fully developed vitro-plants grown within an in vitro environment. Hence, the last stage of the micropropagation process is the acclimatization of the vitro-plants, which are gradually adapted to live in external conditions outside the glass container and the growth room. A protocol has been developed at CICY to acclimate the coconut vitro-plants, and close to 80% survival can be obtained. This protocol is described here.

Scale-Up of Coffea canephora Somatic Embryogenesis in Temporary Immersion System.

Somatic embryogenesis (SE) is a clear example of cellular totipotency. The SE of the genus Coffea has become a model for in vitro propagation for woody species and for the large-scale production of disease-free plants that provide an advantage for modern agriculture. Temporary immersion systems (TIS) are in high demand for the propagation of plants. The success of this type of bioreactor is based on the alternating cycles of immersion of the plant material in the culture medium, usually a few minutes, and the permanence outside the medium of the tissues for several hours. Some bioreactors are very efficient for propagating one species but not another. The efficiency of bioreactors depends on the species, the tissue used to propagate, the species' nutritional needs, the amount of ethylene produced by the tissue, and many more. In this protocol, we show how we produce C. canephora plants that are being taken to the field.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Direct Somatic Embryogenesis in Carica papaya L. Genotypes for Genetic Modification Purposes.

This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5 months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.

Transcriptomic Analysis During the Induction of Somatic Embryogenesis in Coffea canephora.

Omic tools have changed the way of doing research in experimental biology. The somatic embryogenesis (SE) study has not been immune to this benefit. The transcriptomic tools have been used to compare the genes expressed during the induction of SE with the genes expressed in zygotic embryogenesis or to compare the development of the different stages embryos go through. It has also been used to compare the expression of genes during the development of calli from which SE is induced, as well as many other applications. The protocol described here is employed in our laboratory to extract RNA and generate several transcriptomes for the study of SE on Coffea canephora.

Mechanical forces orchestrate the metabolism of the developing oilseed rape embryo.

The initial free expansion of the embryo within a seed is at some point inhibited by its contact with the testa, resulting in its formation of folds and borders. Although less obvious, mechanical forces appear to trigger and accelerate seed maturation. However, the mechanistic basis for this effect remains unclear. Manipulation of the mechanical constraints affecting either the in vivo or in vitro growth of oilseed rape embryos was combined with analytical approaches, including magnetic resonance imaging and computer graphic reconstruction, immunolabelling, flow cytometry, transcriptomic, proteomic, lipidomic and metabolomic profiling. Our data implied that, in vivo, the imposition of mechanical restraints impeded the expansion of testa and endosperm, resulting in the embryo's deformation. An acceleration in embryonic development was implied by the cessation of cell proliferation and the stimulation of lipid and protein storage, characteristic of embryo maturation. The underlying molecular signature included elements of cell cycle control, reactive oxygen species metabolism and transcriptional reprogramming, along with allosteric control of glycolytic flux. Constricting the space allowed for the expansion of in vitro grown embryos induced a similar response. The conclusion is that the imposition of mechanical constraints over the growth of the developing oilseed rape embryo provides an important trigger for its maturation.

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos.

Human fertility is suboptimal, partly due to error-prone divisions in early cleavage-stages that result in aneuploidy. Most human pre-implantation are mosaics of euploid and aneuploid cells, however, mosaic embryos with a low proportion of aneuploid cells have a similar likelihood of developing to term as fully euploid embryos. How embryos manage aneuploidy during development is poorly understood. This knowledge is crucial for improving fertility treatments and reducing developmental defects. To explore these mechanisms, we established a new mouse model of chromosome mosaicism to study the fate of aneuploid cells during pre-implantation development. We previously used the Mps1 inhibitor reversine to generate aneuploidy in embryos. Here, we found that treatment with the more specific Mps1 inhibitor AZ3146 induced chromosome segregation defects in pre-implantation embryos, similar to reversine. However, AZ3146-treated embryos showed a higher developmental potential than reversine-treated embryos. Unlike reversine-treated embryos, AZ3146-treated embryos exhibited transient upregulation of Hypoxia Inducible-Factor-1A (HIF1A) and lacked p53 upregulation. Pre-implantation embryos develop in a hypoxic environment in vivo, and hypoxia exposure in vitro reduced DNA damage in response to Mps1 inhibition and increased the proportion of euploid cells in the mosaic epiblast. Inhibiting HIF1A in mosaic embryos also decreased the proportion of aneuploid cells in mosaic embryos. Our work illuminates potential strategies to improve the developmental potential of mosaic embryos.

ABA exerts a promotive effect on the early process of somatic embryogenesis in Quercus aliena Bl.

Quercus aliena, a native Chinese tree species, is significant in industry and landscaping. However, it is traditionally propagated by seeds with many limitations, such as pest infestations, seed yield and quality. Thus, this study firstly introduces a somatic embryogenesis (SE) system for Q. aliena, enhancing its cultivation prospects. Thereinto, the development stage of zygotic embryo had a significant effect on SE, only immature embryos in 10-11 weeks after full bloom (WAF), rich in endogenous abscisic acid (ABA), could induce SE. Exogenous application ABA had positive roles in the early development process of both primary and secondary SE, while its antagonist had opposite roles. Transcriptome analysis showed that transcription regulation occupied the major position. Mfuzz cluster and WGCNA co-expression analysis showed that 24 candidate genes were involved in the SE process. The expression of the 24 genes were also affected by exogenous ABA signals, among which QaLEC2, QaCALS11 and QaSSRP1 occupied the important roles. Additionally, the callose content were also affected by exogenous ABA signals, which had significantly positive correlations with the expression of QaLEC2 and QaCALS11. This study not only established an efficient reproduction system for Q. aliena, but also revealed the difference in embryogenic ability of zygotic embryos from the aspects of transcriptome and endogenous hormone content, and lay a foundation for clarifying the molecular mechanism of SE, and provided a reference for exploring the vital roles of ABA in SE.

Monosymmetros Cephalothoracopagus Tetrabrachius and Tetrapus Piglets with Syndromic Evolution.

Conjoined twins are rare congenital malformations that have been reported in mammals. Two different cases are presented in this study. Case No. 1 features monocephalic, thoracopagus-conjoined twin piglets with anencephaly and palatoschisis of the Pietrain breed, and case No. 2 features monocephalic, thoracopagus conjoined twin piglets with palatoschisis and bifid root tongue of a mixed breed. These cases were examined using post-mortem and computed tomography (CT) examinations. In both cases, the conjoined symmetrical twins had a single head, one neck, and fused thoracic cavities, while the abdominal cavities were separated. Similarly, in both cases, they had four forelimbs and four hindlimbs and duplicated foramen magnum. During CT examination, in case No. 1, severe abnormalities were observed in the skull and vertebral column. In the left twin, occult dysraphism was seen from the C2 vertebra until the end of the vertebral column, and in the right twin, from the C3 vertebra until the end of the state vertebral level. In case No. 2, the oral cavity contained a tongue with a bifid root connected with one hyoid bone, and the soft palate presented a small cleft. During CT examination, the parietal bone and the occipital bones were partially duplicated. This case also presented occult dysraphism, but only in the cervical vertebrae, C1-C6 for the left twin and C1-C5 for the right twin. In both cases, abnormalities of the internal organs were revealed during necropsy. Conjoined twins with multiple congenital anomalies presented here enhance our understanding of the various clinical forms of conjoined cases in veterinary medicine.