In situ Nuclear Matrix preparation in Drosophila melanogaster and its use in studying the components of nuclear architecture

The study of Nuclear Matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol can be extended to larval tissues like salivary glands and imaginal discs with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant and transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture.

Does the number of embryos loaded on a single cryo-carrier affect post-vitrification survival rate?

Background: We aimed to assess whether the survival rate of embryos is influenced by the number of embryos/oocytes loaded on a single cryo-carrier during vitrification. Methods: This was a retrospective study that included 974 patients who underwent thawing of 1896 embryo-warming cycles between September 2016 and January 2020. A distinct analysis was made for cleavage stage embryos (2–10-cell stage) and blastocysts. For vitrification, embryos were placed in a Cryotop™ open device using a SAGE vitrification kit following the manufacturer’s instructions. Warming was carried using a SAGE warming vitrification kit according the manufacturer’s instructions. Results: Total post-vitrification survival rates of embryos at the cleavage stage or blastocyst stage was 94.8%. At the cleavage stage, cryo-preserving three embryos per single cryo-carrier gave the highest full intact embryo survival rate (91.5%) compared with one or two embryo(s) per single cryo-carrier (85.7%, P < 0.0002 and 87.3%, P < 0.004). Conversely, post warmed full intact blastocyst survival rate for two blastocysts was significantly lower compared with one blastocyst (76.7% vs. 87.9%, P < 0.0193) per single cryo-carrier. Conclusion: Post-thawing survival rate following vitrification is affected by the number of embryos per single cryo-carrier undergoing the vitrification equilibration phase, with the optimum number of three cleaved embryos or one blastocyst per single cryo-carrier. Further studies are required to determine the optimum number of cleaved embryos or blastocysts that should be loaded onto a single cryo-carrier vitrification device.

Totipotency continuity from zygote to early blastomeres: a model under revision

The mammalian zygote is a totipotent cell that generates all the cells of a new organism through embryonic development. However, if one asks about the totipotency of blastomeres after one or two zygotic divisions, opinions differ. As it is impossible to determine the individual developmental potency of early blastomeres in an intact embryo, experiments of blastomere isolation were conducted in various species, showing that two-cell blastomeres could give rise to a new organism when sister cells were separated. A mainstream interpretation was that each of the sister mammalian blastomeres was equally totipotent. However, reevaluation of those experiments raised some doubts about the real prevalence of cases in which this interpretation could truly be validated. We compiled experiments that tested the individual developmental potency of early mammalian blastomeres in a cell-autonomous way (i.e. excluding nuclear transfer and chimera production). We then confronted the developmental abilities with reported molecular differences between sister blastomeres. The reevaluated observations were at odds with the mainstream view: A viable two-cell embryo can already include one non-totipotent blastomere. We were, thus, led to propose a revised model for totipotency continuity based on the construction of the zygote as a mosaic, which accounts for differential inheritance of totipotency-relevant components between sister blastomeres. This takes place with no preordained mechanisms that would ensure a reproducible partition. This model, which is compatible with the body of data on regulative properties of mammalian early embryos, aims at tempering the rigid interpretation that discounted maternal constraints on totipotency.

Imaging, Visualization, and Computation in Developmental Biology

Embryonic development is highly complex and dynamic, requiring the coordination of numerous molecular and cellular events at precise times and places. Advances in imaging technology have made it possible to follow developmental processes at cellular, tissue, and organ levels over time as they take place in the intact embryo. Parallel innovations of in vivo probes permit imaging to report on molecular, physiological, and anatomical events of embryogenesis, but the resulting multidimensional data sets pose significant challenges for extracting knowledge. In this review, we discuss recent and emerging advances in imaging technologies, in vivo labeling, and data processing that offer the greatest potential for jointly deciphering the intricate cellular dynamics and the underlying molecular mechanisms. Our discussion of the emerging area of “image-omics” highlights both the challenges of data analysis and the promise of more fully embracing computation and data science for rapidly advancing our understanding of biology.

xNgn2 induces expression of predominantly sensory neuron markers in Xenopus whole embryo ectoderm but induces mixed subtype expression in isolated ectoderm explants

Proneural basic-helix-loop-helix (bHLH) proteins, such as Neurogenin2 (Ngn2) and Ascl1, are critical regulators at the onset of neuronal differentiation. Endogenously they have largely complementary expression patterns, and have conserved roles in the specification of distinct neuronal subtypes. In Xenopus embryos, xNgn2 is the master regulator of primary neurogenesis forming sensory, inter- and motor neurons within the neural plate, while xAscl1 is the master regulator of autonomic neurogenesis, forming noradrenergic neurons in the antero-ventral region of the embryo. Here we characterise neuronal subtype identity of neurons induced by xNgn2 in the ectoderm of whole Xenopus embryos in comparison with xAscl1, and in ectodermal “animal cap” explants. We find that the transcriptional cascades mediating primary and autonomic neuron formation are distinct, and while xNgn2 and xAscl1 can upregulate genes associated with a non-endogenous cascade, this expression is spatially restricted within the embryo. xNgn2 is more potent than xAscl1 at inducing primary neurogenesis as assayed by neural-β-tubulin. In ectoderm of the intact embryo, these induced primary neurons have sensory characteristics with no upregulation of motor neuron markers. In contrast, xNgn2 is able to up-regulate both sensory and motor neuron markers in naïve ectoderm of animal cap explants, suggesting a non-permissive environment for motor identity in the patterned ectoderm of the whole embryo.

110 PREGNANCY RATES OF BOVINE EMBRYOS AND HEMI-EMBRYOS TRANSFERENCE

Embryo transfer (ET) in cattle speeds up genetic gains, but its use is limited by cost-benefit analysis. In addition other techniques can be developed, such as sexing and bipartition of embryos. An alternative to improve the economic viability of ET in cattle would be to bipartition embryos in order to increase the yield of the technique. The aim of this study was to investigate the operational viability of embryo bipartition technique in an ET program in cattle and to study aspects related to the viability and development of bovine hemi-embryos (HE) compared with intact ones. The embryos were collected by nonsurgical technique 7 days after the onset of oestrus. Viable structures (130) from 49 embryo collections from 25 Aberdeen and Simmental donor cows and heifers were used. Only embryos in compact morula, early blastocyst and blastocyst stage, with a morphological range from excellent to good grade (IETS Grade 1), were split. The embryos, split (78) or not (52), were transferred into the uterine horn ipsilateral to the corpus luteum of the recipients 7 days after oestrus. The treatment groups evaluated were T1 (intact embryo, n = 52), T2 (1 HE, n = 27) and T3 (2 HE, n = 51). Crossbred heifers were used as recipients and pregnancy diagnosis was done at 60 to 80 days of gestation. The embryos of T1, T2 and T3 were classified morphologically as excellent or good and by developmental stage as morula, early blastocyst, or blastocyst, distributed as follows: T1: 30, 22 and 16, 17 and 19; T2: 15, 12 and 7, 9 and 11; and T3: 29, 22 and 15, 15 and 21, respectively. The birth rate per original embryos was greater for T2 than for T1 and T3 (Table 1). The pregnancy rates for excellent and good embryos and morulae, early blastocysts and blastocysts were not different (P > 0.05). The T1, T2 and T3 twin births were 0, 1 and 5, respectively. It is concluded that embryo bipartition technique applied in a commercial ET program is a viable operational technique. Table 1.Pregnancy at 60 to 80 days and birth rate per original embryos used Supported by CAPES, CNPq, FAPEMIG and BIOTRAN.

283 TRANSCRIPTIONAL ANALYSIS OF BIOPSIES DERIVED FROM IN VITRO-PRODUCED BOVINE BLASTOCYSTS IN RELATION TO PREGNANCY SUCCESS AFTER TRANSFER TO RECIPIENTS

Early embryonic mortality is a recognized cause of reproductive failure in cattle leading to the loss of a large number of potential calves, retarded genetic progress, and significant loss of money and time in rebreeding cows. The purpose of this work was to address the relationship between the transcriptional profile of embryos, using the microarray technique, and pregnancy success based on blastocyst biopsies taken prior to transfer to recipients. Biopsies (3 to 40% of the intact embryo) were taken from IVP Day 7 blastocysts (n = 98) and 60 to 70% was transferred to recipients (2-year-old Simmental heifers) after re-expansion. Based on the success of pregnancy, biopsies were pooled in 3 groups: those resulting in no pregnancy (G1), resorption (G2), and calf delivery (G3). Gene expression analysis of these groups of biopsies was performed using a home-made bovine pre-implantation-specific array (with 219 clones) and bovine cDNA array (BlueChip) (with 2000 clones) (Sirard et al. 2005 Reprod. Fertil. Dev. 17, 47–57). Approximately 3 �g of amplified RNA from pooled biopsies (10 biopsies each) was used as a template in reverse transcription reactions incorporating amino-modified dUTPs into labeled cDNA using the CyScribeTM post-labelling kit (Amersham Bioscience, Freiburg, Germany). The synthesized cDNAs from all 3 groups were differentially labeled using Cy3 and Cy5 dyes. Slides were scanned using a GenePix 4000B scanner and images were analyzed using GenePix Pro Version 4.0 software (Axon Instruments, Union City, CA, USA). Data were analyzed using Significant Analysis for Microarray (SAM) software (http//www.stst.stanford.edu/tips/SAM). Quantitative real-time PCR was used to confirm the differentially expressed genes revealed by microarray experiments. Results revealed that 52 genes were differentially regulated between G1 and G3, and 58 genes were differentially regulated between G2 and G3. Biopsies resulting in calf delivery were enriched with genes necessary for implantation (COX-2 and CDX2), signal transduction (PLAU), polyamine biosynthesis (ODC1), response to oxidative stress (TXN), growth factor (BMP15), and placenta-specific 8 (PLAC8). Biopsies that ended up with resorption were enriched with transcripts involving protein phosphorylation (KRT8) plasma membrane (OCLN), and glucose metabolism (PGK1, AKR1B1). Biopsies resulting in no pregnancy were enriched with transcripts involving inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes that determine the fate of the embryo after transfer.

Early development of nematode embryos: differences and similarities

To determine whether embryogenesis of Caenorhabditis elegans is typical for nematodes in general, we started to analyse in comparison several aspects of development in various nematode species. The differences we observed can be subdivided into two classes, those visible in the intact embryo and those requiring experimental interference. Particularly obvious differences of both types were revealed between C. elegans (Rhabditidae) and Acrobeloides nanus (Cephalobidae). Not only does the spatial and temporal pattern of early events differ but also that of intercellular communication and cell specification. Our data suggest that some developmental variations are characteristic for certain nematode groups and therefore may be useful as phylogenetic markers. In contrast, we detected little evidence so far for environmental influence on early developmental processes. Pour déterminer dans quelle mesure l’embryogenèse de Caenorhabditis elegans est une caractéristique générale des nématodes, nous avons commencé l’analyse de plusieurs aspects du développement chez différentes espèces de nématodes. Les différences observées peuvent être divisées en deux catégories: celles observables chez l’embryon intact et celles nécessitant une intervention expérimentale. En particulier, des différences nettes entre les deux catégories ont été mises en évidence chez C. elegans (Rhabditidae) et Acrobeloides nanus (Cephalobidae). Diffèrent non seulement le schéma spatio-temporel des évènements précoces, mais également la communication intercellulaire et la différenciation cellulaire. Nos données suggèrent que certaines variations du développement sont caractéristiques de certains groupes de nématodes et pourraient donc être utiles comme marqueurs phylogénétiques. A contrario, une influence de l’environnement sur les processus précoces du développement n’a pas, jusqu’à présent, été détectée.

Establishment of gut fate in the E lineage of C. elegans: the roles of lineage-dependent mechanisms and cell interactions

The gut of C. elegans derives from all the progeny of the E blastomere, a cell of the eight cell stage. Previous work has shown that gut specification requires an induction during the four cell stage (Goldstein, B. (1992) Nature 357, 255–257). Blastomere isolation and recombination experiments were done to determine which parts of the embryo can respond to gut induction. Normally only the posterior side of the EMS blastomere contacts the inducing cell, P2. When P2 was instead placed in a random position on an isolated EMS, gut consistently differentiated from the daughter of EMS contacting P2, indicating that any side of EMS can respond to gut induction. Additionally, moving P2 around to the opposite side of EMS in an otherwise intact embryo caused EMS's two daughter cells to switch lineage timings, and gut to differentiate from the descendents of what normally would be the MS blastomere. The other cells of the four cell stage, ABa, ABp, and P2, did not form gut when placed in contact with the inducer. To determine whether any other inductions are involved in gut specification, timed blastomere isolations were done at the two and eight cell stages. In the absence of cell contact at the two cell stage, segregation of gut fate proceeded normally at both the two and four cell stages. Gut fate also segregated properly in the absence of cell contact at the eight cell stage. A model is presented for the roles of lineage-dependent mechanisms and cell interactions in establishing gut fate in the E lineage.

Altered expression of spatially regulated embryonic genes in the progeny of separated sea urchin blastomeres

We have examined the importance of the extracellular environment on the ability of separated cells of sea urchin embryos (Strongylocentrotus purpuratus) to carry out patterns of mRNA accumulation and decay characteristic of intact embryos. Embryos were dissociated into individual blastomeres at 16-cell stage and maintained in calcium-free sea water so that daughter cells continuously separated. Levels of eleven different mRNAs in these cells were compared to those in control embryos when the latter reached mesenchyme blastula stage, by which time cells in major regions of the intact embryo have assumed distinctive patterns of message accumulation. Abrogation of interactions among cells resulted in marked differences in accumulation and/or turnover of the individual mRNAs, which are expressed with diverse temporal and spatial patterns of prevalence in intact embryos. In general, separated cells are competent to execute initial events of mRNA accumulation and decay that occur uniformly in most or all blastomeres of the intact embryo and are likely to be regulated by maternal molecules. The ability of separated cells to accumulate mRNAs that appear slightly later in development depends upon the presumptive tissue in which a given mRNA is found in the normal embryo. Messages that normally accumulate in cells at the vegetal pole also accumulate in dissociated cells either at nearly normal levels or at increased levels. In one such case, that of actin CyIIa, which is normally restricted to mesenchyme cells, in situ hybridization demonstrates that the fraction of dissociated cells expressing this message is 4- to 5-fold higher than in the normal embryo. In contrast, separated cells accumulate significant levels of a message expressed uniformly in the early ectoderm but are unable to execute accumulation and decay of different messages that distinguish oral and aboral ectodermal regions. These data are consistent with the idea that interactions among cells in the intact embryo are important for both positive and negative control of expression of different genes that are early indicators of the specification of cell fate.