Early Cell Division Research Articles

Night Monkey Hybrids Exhibit De Novo Genomic and Karyotypic Alterations: The First Such Case in Primates.

Using molecular chromosomal analyses, we discovered night monkey hybrids produced in captivity from matings between a female Aotus azarae boliviensis (2n = 50) and a male Aotus lemurinus griseimembra (2n = 53). The parents produced seven offspring in total, including one male and six females—a pattern consistent with Haldane’s rule. Chromosomal studies were conducted on four of the hybrid offspring. Two of them showed relatively “simple” mixture karyotypes, including different chromosome numbers (2n = 51, 52), which were formed because of a heteromorphic autosome pair in the father (n = 26, 27). The other two hybrid monkeys exhibited de novo genomic and karyotypic alterations. Detailed analysis of the alterations revealed that one individual carried a mixture karyotype of the two parental species and an X chromosome trisomy (53,XXX). The second individual displayed trisomy of chromosome 18 (52,XX,+18) and a reciprocal translocation between autosomes 21 and 23 (52,XX,+18,t(21;23)). Interestingly, the second monkey exhibited mosaicism among blood cells (mos52,XX,+18[87]/52,XX,+18,t(21;23)[85]), but only a single karyotype (52,XX,+18) in skin fibroblast cells. The X- and 18-trisomies were derived from a doubling of the mother’s chromosomes in early embryonic cell division, and the reciprocal translocation likely developed in the bone marrow of the offspring, considering that it was observed only in blood cells. Such occurrence of trisomies in hybrid individuals is a unique finding in placental mammals.

Abnormal cleavage patterns in embryos are associated with aneuploidy and poor morphology scores

Morphokinetic parameters as measured through time-lapse imaging may predict embryonic developmental potential. While there are several studies on the timing of early cell divisions, there are limited data on abnormal cleavage (AC) events and their relationship to embryo quality. An AC is the division from a mother blastomere cell to three daughter cells. This is thought to be secondary to abnormal mitotic spindle assembly, leading to an abnormal segregation of chromosomes. Previous studies report that embryos exhibiting ACs have decreased blastulation and worse implantation rates. In our study, we sought to determine if ACs are associated with aneuploidy. We also evaluated the relationship between ACs and developmental potential as well as day 3 and day 5 morphologic scoring. Our primary objective was to determine if ACs are related to ploidy status. Secondary objectives were to determine if ACs are correlated with blastulation rates and with embryo morphologic scores at the cleavage and blastocyst stages. Previously cryopreserved human embryos donated for research were thawed and cultured in the Miri Time-lapse incubator for 6 days. A staff embryologist recorded standard time-lapse parameters and day 3 and 5 morphology assessments. Trophectoderm biopsies were performed on embryos that grew to blastocysts on day 5 or 6. Otherwise whole embryos were sent for preimplantation genetic screening (PGS). Next generation sequencing was used to determine embryo ploidy status. Morphologic grading of embryos was performed per Society of Assisted Reproductive Technology guidelines for cleavage stage embryos. We used methods for blastocyst morphologic grading described by Gardner, et al with minor modifications. Statistics were performed using Chi-square, Fisher’s Exact, ANOVA, and logistic regression. 50 embryos were included our analysis. 32/50 (64%) of thawed embryos grew to the blastocyst stage. 35/50 (70%) of embryos were euploid. ACs were visualized in 15/50 (30%) embryos. 8/15 (53.3%) of these embryos experienced an AC after the 1st cytokinesis. 7/15 (46.7%) of embryos had ACs after the 2nd cytokinesis. 4/15 (26.7%) embryos had more than 1 AC event. Aneuploid embryos had a significantly higher percentage of AC events (73.3% vs 22.9%, p<0.001). AC events did not reliably predict which embryos grew to the blastocyst stage or those that arrested. Embryos that had at least 1 AC took an average of 10.7 hours longer to develop from a morula to a fully expanded blastocyst compared to those without ACs (β=10.7, p=0.01). Cleavage stage morphology scores did not correlate with ACs. In blastocysts with ACs, there was a higher proportion of poor quality embryos compared with good/fair embryos (66.7% vs 33.3%, p=0.03). Based on our findings, ACs were more likely to occur in aneuploid embryos. While they did not predict blastocyst formation, they were associated with delayed blastulation and with poor blastocyst morphology scores. When selecting embryos for transfer, particularly those not undergoing PGS, those with ACs may have a worse prognosis for achieving a successful pregnancy.

The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus

The assembly of the bacterial cell wall requires synchronization of a multitude of biosynthetic machineries and regulatory networks. The eukaryotic-like serine/threonine kinase PknB has been implicated in coordinating cross-wall formation, autolysis and cell division in Staphylococcus aureus. However, the signal molecule sensed by this kinase remained elusive so far. Here, we provide compelling biochemical evidence that PknB interacts with the ultimate cell wall precursor lipid II, triggering kinase activity. Moreover, we observed crosstalk of PknB with the two component system WalKR and identified the early cell division protein FtsZ as another PknB phosphorylation substrate in S. aureus. In agreement with the implied role in regulation of cell envelope metabolism, we found PknB to preferentially localize to the septum of S. aureus and the PASTA domains to be crucial for recruitment to this site. The data provide a model for the contribution of PknB to control cell wall metabolism and cell division.

Embryonic hypocellularity, blastogenetic malformations, and fetal growth restriction.

An association between congenital malformations and fetal growth restriction (FGR) can be largely explained by a relationship with early embryonic hypocellularity. The malformations include the VACTERL association, which is exceptional as a Mendelian syndrome, but is commonly associated with monozygotic twinning, maternal diabetes, and some forms of aneuploidy, all characterized by a small embryo early in development. Parsimony suggests that these different links to VACTERL are related to the hypocellularity as a single common factor, rather than as an expression of three independent pathogenetic processes. A distinct non-genetic pathogenesis is further supported by increased frequencies in the same conditions of a single umbilical artery (SUA), which is also unusual in Mendelian disorders. SUA often involves the atrophy of one artery, which may be facilitated by altered hemodynamics in a smaller embryo, providing a direct link to hypocellularity. Hypocellularity may also explain a possible connection between VACTERL and certain mitochondrial disorders, where reduced energy might slow early cell division and growth, reducing the size of the embryo. © 2016 Wiley Periodicals, Inc.

Embryo selection using time-lapse analysis (Early Embryo Viability Assessment) in conjunction with standard morphology: a prospective two-center pilot study.

Does prospective embryo selection using the results from the Eava Test (Early Embryo Viability Assessment) in combination with standard morphology increase the pregnancy rate of IVF and ICSI patients compared to embryo selection based on morphology only? Embryo selection using the Eeva Test plus standard morphology on Day 3 results in comparable pregnancy rates as conventional morphological embryo selection. Time-lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. The Eeva Test records the development of each embryo with a cell-tracking system and predicts the likelihood (High, Medium or Low) that an embryo will form a blastocyst based on an automated analysis of early cell division timings. This trial was designed as a prospective, observational, two-center pilot study with a propensity matched control group. The analysis involved 280 of 302 enrolled patients who were included in the Eeva Test group in 2013 and 560 control patients who were treated in the years 2011-2013. The majority of transfers (98%) were single embryo transfers. Two academic hospitals (VUmc Amsterdam and UZ Gent) enrolled patients <41 years old, with <3 previous attempts and ≥5 normally fertilized eggs. Propensity matching was used to identify a propensity matched control group from a cohort of 1777 patients based on age, cycle number, oocyte number and number of fertilized oocytes. There was no difference in patient baseline characteristics between the two groups. The ongoing pregnancy rate (OPR) of patients enrolled in the Eeva Test group (34.3%; 96/280) did not differ significantly from the OPR in the propensity matched control group (34.6%, 194/560; P =0.92). However, significantly less top quality embryos (eight-cell embryos with ≤25% fragmentation) were transferred in the Eeva Test group compared to the propensity matched control group (70.4% vs. 82.3%; P<0.001). The transfer of Eeva High and Medium embryos resulted in a significantly higher OPR of 36.8% (89/242) compared to 18.4% (7/38) for Eeva Low embryos (P=0.02). This pilot study is limited by its nonrandomized design with a concurrent and historical control. Our pilot data did not reveal significant differences between time-lapse based and conventional embryo selection. Interestingly, the pregnancy rates were comparable in both groups even though the morphological quality of the transferred embryos was significantly lower in the Eeva Test group compared to the propensity matched control group. A sufficiently powered three-armed randomized controlled trial (RCT) with a solid design should be performed to generate decisive evidence in the future. Progyny Inc., formerly Auxogyn provided the Eeva scopes, software and technical support for this study. The funding sources did neither influence data collection, management, analysis and interpretation of the data, nor the preparation of the manuscript. ClinicalTrials.gov: NCT01671644.

Phosphorylation of Astrin Regulates Its Kinetochore Function

The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.

Tissue homogeneity requires inhibition of unequal gene silencing during development.

Multicellular organisms can generate and maintain homogenous populations of cells that make up individual tissues. However, cellular processes that can disrupt homogeneity and how organisms overcome such disruption are unknown. We found that ∼100-fold differences in expression from a repetitive DNA transgene can occur between intestinal cells in Caenorhabditis elegans These differences are caused by gene silencing in some cells and are actively suppressed by parental and zygotic factors such as the conserved exonuclease ERI-1. If unsuppressed, silencing can spread between some cells in embryos but can be repeat specific and independent of other homologous loci within each cell. Silencing can persist through DNA replication and nuclear divisions, disrupting uniform gene expression in developed animals. Analysis at single-cell resolution suggests that differences between cells arise during early cell divisions upon unequal segregation of an initiator of silencing. Our results suggest that organisms with high repetitive DNA content, which include humans, could use similar developmental mechanisms to achieve and maintain tissue homogeneity.

RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila

The present study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. We found that deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. We found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins.

Functional Interplay of Two Paralogs Encoding SWI/SNF Chromatin-Remodeling Accessory Subunits During Caenorhabditis elegans Development.

SWI/SNF ATP-dependent chromatin-remodeling complexes have been related to several cellular processes such as transcription, regulation of chromosomal stability, and DNA repair. The Caenorhabditis elegans gene ham-3 (also known as swsn-2.1) and its paralog swsn-2.2 encode accessory subunits of SWI/SNF complexes. Using RNA interference (RNAi) assays and diverse alleles we investigated whether ham-3 and swsn-2.2 have different functions during C. elegans development since they encode proteins that are probably mutually exclusive in a given SWI/SNF complex. We found that ham-3 and swsn-2.2 display similar functions in vulva specification, germline development, and intestinal cell proliferation, but have distinct roles in embryonic development. Accordingly, we detected functional redundancy in some developmental processes and demonstrated by RNA sequencing of RNAi-treated L4 animals that ham-3 and swsn-2.2 regulate the expression of a common subset of genes but also have specific targets. Cell lineage analyses in the embryo revealed hyper-proliferation of intestinal cells in ham-3 null mutants whereas swsn-2.2 is required for proper cell divisions. Using a proteomic approach, we identified SWSN-2.2-interacting proteins needed for early cell divisions, such as SAO-1 and ATX-2, and also nuclear envelope proteins such as MEL-28. swsn-2.2 mutants phenocopy mel-28 loss-of-function, and we observed that SWSN-2.2 and MEL-28 colocalize in mitotic and meiotic chromosomes. Moreover, we demonstrated that SWSN-2.2 is required for correct chromosome segregation and nuclear reassembly after mitosis including recruitment of MEL-28 to the nuclear periphery.

Embryo Development and Morphology of the Rocky Mountain Wood Tick (Acari: Ixodidae).

Dermacentor andersoni Stiles embryogenesis was observed using fluorescent and scanning electron microscopy for eggs held under laboratory conditions (25°C and at 93% relative humidity). Early embryonic cell divisions appeared to be synchronous and holoblastic, giving rise to a uniform blastoderm surrounding the yolk. The cells of the blastoderm became concentrated on one side of the embryo, forming the segmented germ band. Distinct opisthosomal and prosomal segment morphologies, which are characteristic of chelicerate embryos, were observed during germ band elongation. Mouth and leg appendages grew from the prosomal segments. As development progressed, the segments were fused into the idiosoma and capitulum of the free-living larval form. An embryo staging system was established based on embryo developmental morphology and the timing of morphogenetic events. The staging system will serve as a basis for future studies directed at understanding morphogenetic mechanisms or for observing the impact of abiotic factors, such as temperature or humidity, on tick development.

Human embryos commonly form abnormal nuclei during development: a mechanism of DNA damage, embryonic aneuploidy, and developmental arrest.

What is the prevalence and developmental significance of morphologic nuclear abnormalities in human preimplantation embryos? Nuclear abnormalities are commonly found in human IVF embryos and are associated with DNA damage, aneuploidy, and decreased developmental potential. Early human embryonic development is complicated by genomic errors that occur after fertilization. The appearance of extra-nuclear DNA, which has been observed in IVF, may be a result of such errors. However, the mechanism by which abnormal nuclei form and the impact on DNA integrity and embryonic development is not understood. Cryopreserved human cleavage-stage embryos (n = 150) and cryopreserved blastocysts (n = 105) from clinical IVF cycles performed between 1997 and 2008 were donated for research. Fresh embryos (n = 60) of poor quality that were slated for discard were also used. Immunohistochemical, microscopic and cytogenetic analyses at different developmental stages and morphologic grades were performed. Embryos were fixed and stained for DNA, centromeres, mitotic activity and DNA damage and imaged using confocal microscopy. Rates of abnormal nuclear formation were compared between morphologically normal cleavage-stage embryos, morphologically normal blastocysts, and poor quality embryos. To control for clinical and IVF history of oocytes donors, and quality of frozen embryos within our sample, cleavage-stage embryos (n = 52) were thawed and fixed at different stages of development and then analyzed microscopically. Cleavage-stage embryos (n = 9) were thawed and all blastomeres (n = 62) were disaggregated, imaged and analyzed for karyotype. Correlations were made between microscopic and cytogenetic findings of individual blastomeres and whole embryos. The frequency of microscopic nuclear abnormalities was lower in blastocysts (5%; 177/3737 cells) than in cleavage-stage embryos (16%, 103/640 blastomeres, P < 0.05) and highest in arrested embryos (65%; 44/68 blastomeres, P < 0.05). DNA damage was significantly higher in cells with microscopic nuclear abnormalities (γH2AX (phosphorylated (Ser139) histone H2A.X): 87.1%, 74/85; replication protein A: 72.9%, 62/85) relative to cells with normal nuclear morphology (γH2AX: 9.3%, 60/642; RPA: 5.6%, 36/642) (P < 0.05). Blastomeres containing nuclear abnormalities were strongly associated with aneuploidy (Fisher exact test, two-tailed, P < 0.01). The embryos used were de-identified, and the clinical and IVF history was unknown. This study explores a mechanism of abnormal embryonic development post-fertilization. While most of the current data have explored abnormal meiotic chromosome segregation in oocytes as a primary mechanism of reproductive failure, abnormal nuclear formation during early mitotic cell division in IVF embryos also plays a significant role. The detection of abnormal nuclear formation may have clinical application in noninvasive embryo selection during IVF. The study was supported by Columbia University and the New York Stem Cell Foundation. Authors declare no competing interest.

Telomere dynamics during aging in polygenic left ventricular hypertrophy.

Short telomeres are associated with increased risk of cardiovascular disease. Here we studied cardiomyocyte telomere length at key ages during the ontogeny of cardiac hypertrophy and failure in the hypertrophic heart rat (HHR) and compared these with the normal heart rat (NHR) control strain. Key ages corresponded with the pathophysiological sequence beginning with fewer cardiomyocytes (2 days), leading to left ventricular hypertrophy (LVH) (13 wk) and subsequently progression to heart failure (38 wk). We measured telomere length, tissue activity of telomerase, mRNA levels of telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc), and expression of the telomeric regulator microRNA miR-34a. Cardiac telomere length was longer in the HHR compared with the control strain at 2 days and 38 wk, but shorter at 13 wk. Neonatal HHR had higher cardiac telomerase activity and expression of Tert and miR-34a. Telomerase activity was not different at 13 or 38 wk. Tert mRNA and Terc RNA were overexpressed at 38 wk, while miR-34a was overexpressed at 13 wk but downregulated at 38 wk. Circulating leukocytes were strongly correlated with cardiac telomere length in the HHR only. The longer neonatal telomeres in HHR are likely to reflect fewer fetal and early postnatal cardiomyocyte cell divisions and explain the reduced total cardiomyocyte complement that predisposes to later hypertrophy and failure. Although shorter telomeres were a feature of cardiac hypertrophy at 13 wk, they were not present at the progression to heart failure at 38 wk.

Redox and hormone profiling of a Nicotiana tabacum dedifferentiated protoplast culture suggests a role for a cytokinin and gibberellin in plant totipotency

The ability of plant tissues to retain totipotency despite being fully differentiated has been documented for decades. The transition from mature plant tissue to rejuvenated tissue first requires dedifferentiation of mature tissue, followed by rejuvenation (re-entry into the cell cycle) and somatic embryogenesis. We used a Nicotiana tabacum protoplast-based culture system to elucidate the role played by redox and phytohormone networks during the process of dedifferentiation and rejuvenation. Classical markers of redox homeostasis were measured during the rejuvenation process and lipid peroxidation is proposed as the best marker for indicating recovery of cells from oxidative stress sustained during the process of protoplast preparation and culture, prior to rejuvenation. A transient increase at 24 h after culture (HAC) in levels of a cytokinin riboside, iPA, suggests a putative novel function in initiating a stem-cell niche in an auxin dependent manner. A sharp rise at 72 HAC of gibberellin GA4, furthermore suggests a function for this hormone during the process of rejuvenation. These two key findings are consistent with previously described plant models for lateral root developmental. Therein, iPA could be involved in ‘stem-cell-niche’ initiation. Subsequently, GA4 could be involved in rapidly suppressing this initiation step following the earliest cell divisions, thereby enabling the establishment of this ‘niche’ into a callus-like tissue.

Vertebrate Reproduction.

Vertebrate reproduction requires a myriad of precisely orchestrated events-in particular, the maternal production of oocytes, the paternal production of sperm, successful fertilization, and initiation of early embryonic cell divisions. These processes are governed by a host of signaling pathways. Protein kinase and phosphatase signaling pathways involving Mos, CDK1, RSK, and PP2A regulate meiosis during maturation of the oocyte. Steroid signals-specifically testosterone-regulate spermatogenesis, as does signaling by G-protein-coupled hormone receptors. Finally, calcium signaling is essential for both sperm motility and fertilization. Altogether, this signaling symphony ensures the production of viable offspring, offering a chance of genetic immortality.

Evaluation of embryo implantation potential after vitrified and warmed single blastocyst transfer per embryo categorization using the Eeva™ test, an automated time-lapse quantitative analysis

The Eeva Test provides a set of categories predicting embryo developmental potential based on an automated analysis of early cell division timings. The aim of this study was to evaluate the percentage of embryos vitrified at the blastocyst stage and to compare the implantation rate of the vitrified and warmed blastocysts in each Eeva Test category (HIGH, MEDIUM, and LOW), in a group of supernumerary embryos after single embryo transfer (SET) on day 3.

Splitting the cell, building the organism: Mechanisms of cell division in metazoan embryos.

The unicellular metazoan zygote undergoes a series of cell divisions that are central to its development into an embryo. Differentiation of embryonic cells leads eventually to the development of a functional adult. Fate specification of pluripotent embryonic cells occurs during the early embryonic cleavage divisions in several animals. Early development is characterized by well-known stages of embryogenesis documented across animals--morulation, blastulation, and morphogenetic processes such as gastrulation, all of which contribute to differentiation and tissue specification. Despite this broad conservation, there exist clearly discernible morphological and functional differences across early embryonic stages in metazoans. Variations in the mitotic mechanisms of early embryonic cell divisions play key roles in governing these gross differences that eventually encode developmental patterns. In this review, we discuss molecular mechanisms of both karyokinesis (nuclear division) and cytokinesis (cytoplasmic separation) during early embryonic divisions. We outline the broadly conserved molecular pathways that operate in these two stages in early embryonic mitoses. In addition, we highlight mechanistic variations in these two stages across different organisms. We finally discuss outstanding questions of interest, answers to which would illuminate the role of divergent mitotic mechanisms in shaping early animal embryogenesis.

The KNOTTED-like genes of peach (Prunus persica L. Batsch) are differentially expressed during drupe growth and the class 1 KNOPE1 contributes to mesocarp development

The KNOTTED-like transcription factors (KNOX) contribute to plant organ development. The expression patterns of peach KNOX genes showed that the class 1 members act precociously (S1–S2 stages) and differentially during drupe growth. Specifically, the transcription of KNOPE1 and 6 decreased from early (cell division) to late (cell expansion) S1 sub-stages, whilst that of STMlike1, 2, KNOPE2, 2.1 ceased at early S1. The KNOPE1 role in mesocarp was further addressed by studying the mRNA localization in the pulp cells and vascular net at early and late S1. The message signal was first diffuse in parenchymatous cells and then confined to hypodermal cell layers, showing that the gene down-tuning accompanied cell expansion. As for bundles, the mRNA mainly featured in the procambium/phloem of collateral open types and subsequently in the phloem side of complex structures (converging bundles, ducts). The KNOPE1 overexpression in Arabidopsis caused fruit shortening, decrease of mesocarp cell size, diminution of vascular lignification together with the repression of the major gibberellin synthesis genes AtGA20ox1 and AtGA3ox1. Negative correlation between the expression of KNOPE1 and PpGA3ox1 was observed in four cultivars at S1, suggesting that the KNOPE1 repression of PpGA3ox1 may regulate mesocarp differentiation by acting on gibberellin homeostasis.

Inhibition of SOCE disrupts cytokinesis in zebrafish embryos via inhibition of cleavage furrow deepening.

During the first few cell division cycles in zebrafish, distinct Ca(2+) transients are localized to the early embryonic cleavage furrows, where they accompany (and are required for) furrow positioning, propagation, deepening and apposition. It has previously been shown that the endoplasmic reticulum (ER) acts as the primary store for generating these Ca(2+) transients via release through inositol 1,4,5-trisphosphate receptors (IP 3Rs). We hypothesised that maintaining the elevated levels of intracellular Ca(2+) required for deepening and apposition of the cleavage furrows in these large eggs might result in the depletion of the available ER Ca(2+) store, thus the role of store-operated Ca(2+) entry (SOCE) was examined. Newly fertilized, dechorionated embryos were incubated with various SOCE inhibitors, starting just prior to the onset of the first cell division cycle. The effect of these inhibitors on mitosis, furrow positioning, propagation, deepening and apposition, and the generation of the cytokinetic Ca(2+) transients was determined. Treatment with 2-APB or SKF 96365 had no major effect on mitosis, furrow positioning or propagation, but inhibited furrow deepening resulting in regression of the cleavage furrow. Both of these inhibitors also blocked the furrowing Ca(2+) transient, with SKF 96365 having a more profound inhibitory effect than 2-APB. In zebrafish, SOCE does not appear to be required for mitosis or the early stages of cytokinesis during the early embryonic cell division cycles, but it does appear to be essential for maintaining the elevated levels of [Ca(2+)]i for the extended periods that are required during furrow deepening and daughter cell apposition.

Ultrastructural and structural characterization of zygotes and embryos during development in Sargassum cymosum (Phaeophyceae, Fucales).

This study investigates the pattern and performance of cellular structures during the early development of zygotes and embryos of Sargassum cymosum. The early development S. cymosum germlings has already been characterized and compared with the pattern of development established for all fucoid algae, in which the zygote remains attached to the receptacle by mucilage during the establishment of polarity and early cell division. As in the algae Fucus and Silvetia, the first division is transverse across the longer axis of the zygote of S. cymosum. However, the cell that will give rise to the rhizoids is not determined in the first division; rather, the formation of this cell occurs with the second division, forming a small cell in the embryo shaded site. Stabilizing polarity during the process of forming a multicellular embryo occurs rapidly. During development, significant cytoplasmic alterations take place. Initially, the cytoplasm shows large clusters of phenolic compounds located in specific parts, but later, in the course of development, these compounds are dispersed in the cytoplasm, although a significant amount remains confined to the nucleus. Moreover, to produce more zygotes and higher growth rates for the germlings, the best conditions found for the species S. cymosum were 22 and 26 °C, respectively.

TRANSLOCASE OF THE INNER MEMBRANE9 and 10 Are Essential for Maintaining Mitochondrial Function during Early Embryo Cell and Endosperm Free Nucleus Divisions in Arabidopsis

In the life cycle of flowering plants, the sporophytic generation takes up most of the time and plays a dominant role in influencing plant growth and development. The embryo cell and endosperm free nucleus divisions establish the critical initiation phase of early sporophyte development, which forms mature seeds through a series of cell growth and differentiation events. Here, we report on the biological functions of two Arabidopsis (Arabidopsis thaliana) mitochondrial proteins, TRANSLOCASE OF THE INNER MEMBRANE9 (TIM9) and TIM10. We found that dysfunction of either AtTIM9 or AtTIM10 led to an early sporophyte-lethal phenotype; the embryo and endosperm both arrest division when the embryo proper developed to 16 to 32 cells. The abortion of tim9-1 and tim10 embryos at the 16/32-cell stage was caused by the loss of cell viability and the cessation of division in the embryo proper region, and this inactivation was due to the collapse of the mitochondrial structure and activity. Our characterization of tim9-1 and tim10 showed that mitochondrial membrane permeability increased and that cytochrome c was released from mitochondria into the cytoplasm in the 16/32-cell embryo proper, indicating that mitochondrial dysfunction occurred in the early sporophytic cells, and thus caused the initiation of a necrosis-like programmed cell death, which was further proved by the evidence of reactive oxygen species and DNA fragmentation tests. Consequently, we verified that AtTIM9 and AtTIM10 are nonredundantly essential for maintaining the mitochondrial function of early embryo proper cells and endosperm-free nuclei; these proteins play critically important roles during sporophyte initiation and development in Arabidopsis.