The CNS of the protovertebrate Ciona intestinalis contains a single cluster of dopaminergic (DA) neurons, the coronet cells, which have been likened to the hypothalamus of vertebrates. Whole-embryo single-cell RNA sequencing (RNA-seq) assays identified Ptf1a as the most strongly expressed cell-specific transcription factor (TF) in DA/coronet cells. Knockdown of Ptf1a activity results in their loss, while misexpression results in the appearance of supernumerary DA/coronet cells. Photoreceptor cells and ependymal cells are the most susceptible to transformation, and both cell types express high levels of Meis Coexpression of both Ptf1a and Meis caused the wholesale transformation of the entire CNS into DA/coronet cells. We therefore suggest that the reiterative use of functional manipulations and single-cell RNA-seq assays is an effective means for the identification of regulatory cocktails underlying the specification of specific cell identities.

The development of all vertebrate embryos requires the establishment of a three-dimensional coordinate system in order to pattern embryonic structures and create the complex shape of the adult organism. During the process of gastrulation, the three primary germ layers are created under the guidance of numerous signaling pathways, allowing cells to communicate during development. Cell-cell communication, mediated by receptors of the Notch family, has been shown to be involved in mediating diverse cellular behaviors during development and has been implicated in the regulation of cell fate decisions in both vertebrate and invertebrate organisms. In order to investigate a role for Notch signaling during boundary formation between the mesoderm and endoderm during gastrulation, we manipulated Notch signaling in gastrula stage embryos and examined gene expression in resultant tissues and organs. Our findings demonstrate a much broader role for Notch signaling during germ layer determination than previously reported in a vertebrate organism. Activation of the Notch pathway, specifically in gastrula stage embryos, results in a dramatic decrease in the expression of genes necessary to create many different types of mesodermal tissues while causing a dramatic expansion of endodermal tissue markers. Conversely, temporally controlled suppression of this pathway results in a loss of endodermal cell types and an expansion of molecular markers of mesoderm. Thus, our data are consistent with and significantly extend the implications of prior observations suggesting roles for Notch signaling during germ layer formation and establish an evolutionarily conserved role for Notch signaling in mediating mesoderm-endoderm boundaries during early vertebrate development.

In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem), cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

Finding an effective muscle regeneration technique is a priority for regenerative medicine. It is known that the key factors determining tissue formation include cells, capable of proliferating and/or differentiating, a niche (surface) allowing their colonization and growth factors. The interaction between these factors, especially between the surface of the artificial niche and growth factors, is not entirely clear. Moreover, it seems that the use of a complex of complementary growth factors instead of a few strictly defined ones could increase the effectiveness of tissue maturation, including muscle tissue. In this study, we evaluated whether graphene oxide (GO) nanofilm, chicken embryo muscle extract (CEME), and GO combined with CEME would affect the differentiation and functional maturation of muscle precursor cells, as well as the ability to spontaneously contract a pseudo-tissue muscle. CEME was extracted on day 18 of embryogenesis. Muscle cells obtained from an 8-day-old chicken embryo limb bud were treated with GO and CEME. Cell morphology and differentiation were observed using different microscopy methods. Cytotoxicity and viability of cells were measured by lactate dehydrogenase and Vybrant Cell Proliferation assays. Gene expression of myogenic regulatory genes was measured by Real-Time PCR. Our results demonstrate that CEME, independent of the culture surface, was the main factor influencing the intense differentiation of muscle progenitor cells. The present results, for the first time, clearly demonstrated that the cultured tissue-like structure was capable of inducing contractions without externally applied impulses. It has been indicated that a small amount of CEME in media (about 1%) allows the culture of pseudo-tissue muscle capable of spontaneous contraction. The study showed that the graphene oxide may be used as a niche for differentiating muscle cells, but the decisive influence on the maturation of muscle tissue, especially muscle contractions, depends on the complexity of the applied growth factors.

The Auxin Binding Protein1 (ABP1) has been identified based on its ability to bind auxin with high affinity and studied for a long time as a prime candidate for the extracellular auxin receptor responsible for mediating in particular the fast non-transcriptional auxin responses. However, the contradiction between the embryo-lethal phenotypes of the originally described Arabidopsis T-DNA insertional knock-out alleles ( abp1-1 and abp1-1s) and the wild type-like phenotypes of other recently described loss-of-function alleles ( abp1-c1 and abp1-TD1) questions the biological importance of ABP1 and relevance of the previous genetic studies. Here we show that there is no hidden copy of the ABP1 gene in the Arabidopsis genome but the embryo-lethal phenotypes of abp1-1 and abp1-1s alleles are very similar to the knock-out phenotypes of the neighboring gene, BELAYA SMERT ( BSM). Furthermore, the allelic complementation test between bsm and abp1 alleles shows that the embryo-lethality in the abp1-1 and abp1-1s alleles is caused by the off-target disruption of the BSM locus by the T-DNA insertions. This clarifies the controversy of different phenotypes among published abp1 knock-out alleles and asks for reflections on the developmental role of ABP1.

Early mouse embryos have an atypical translational machinery that consists of cytoplasmic lattices and is poorly competent for translation. Hence, the impact of transcriptomic changes on the operational level of proteins is predicted to be relatively modest. To investigate this, we performed liquid chromatography-tandem mass spectrometry and mRNA sequencing at seven developmental stages, from the mature oocyte to the blastocyst, and independently validated our data by immunofluorescence and qPCR. We detected and quantified 6,550 proteins and 20,535 protein-coding transcripts. In contrast to the transcriptome - where changes occur early, mostly at the 2-cell stage - our data indicate that the most substantial changes in the proteome take place towards later stages, between the morula and blastocyst. We also found little to no concordance between the changes in protein and transcript levels, especially for early stages, but observed that the concordance increased towards the morula and blastocyst, as did the number of free ribosomes. These results are consistent with the cytoplasmic lattice-to-free ribosome transition being a key mediator of developmental regulation. Finally, we show how these data can be used to appraise the strengths and limitations of mRNA-based studies of pre-implantation development and expand on the list of known developmental markers.

Only a few microbial studies have conducted in IVF (in vitro fertilization), showing the high-variety bacterial contamination of IVF culture media to cause damage to or even loss of cultured oocytes and embryos. We aimed to determine the prevalence and counts of bacteria in IVF samples, and to associate them with clinical outcome.

The five-membered SET and MYND domain-containing lysine methyltransferase (SMYD) family plays pivotal roles in development and differentiation. Initially characterized within the cardiovascular system, one such member, SMYD2, has been implicated in transcriptional and apoptotic regulation of hematopoiesis. Deletion of Smyd2 in adult mouse Hemaopoietic Stem Cells (HSC) using an interferon-inducible mx1-Cre-mediated conditional knockout (CKO) led to HSC reduction via both apoptosis and transcriptional deficiencies. Since HSC are specified from hemogenic endothelial (HE) cells in the dorsal aorta (DA), we sought to determine whether the flaw in HSC originated embryologically from this site. Toward this end, we performed deletion with vav-Cre mice, which is active in all hematopoietic and endothelial tissues from E10.5 embryonic life onward. Unexpectedly, we observed no defects in the embryo, other than apoptotic loss of definite HSC, whereas adult hematopoietic populations downstream were unaffected. These results further establish the importance of SMYD2 in antiapoptotic gene control of gene expression from the embryo to the adult.

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.

Early pregnancy loss is a major clinical concern in animal and human reproduction, which is largely influenced by embryo implantation. The importance of methionine for embryo implantation is widely neglected.

Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3'-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.

The transcription factor Barhl2 is an antiproneural transcription factor with roles in neuronal differentiation. The functions of its homologue in Drosophila development are better understood than its functions in mammalian brain development. Existing evidence suggests that its expression in the embryonic forebrain of the mouse is regional and may complement that of another transcription factor that is important for forebrain development, Pax6. The aim of this study is to provide a more detailed description of the Barhl2 expression pattern in the embryonic forebrain than is currently available, to relate its expression domains to those of Pax6 and to examine the effects of Pax6 loss on Barhl2 expression.

The embryo produced by in vitro oocyte maturation, fertilization, and embryonic development is an important resource for genetic improvement and has the potential to improve female fertility and to be programmed to produce offspring with superior ability for health and production. The cultured embryo is also an important component of several realized and potential technologies such as gene editing, somatic cell nuclear cloning, stem cell technologies and gamete generation in vitro. Full realization of the opportunities afforded by the in vitro-produced embryo will require overcoming some technical obstacles to cost-effective implementation of an embryo transfer program. Among the research goals for improving the penetration of embryo transfer in the cattle industry are development of methods to increase the supply of oocytes from genetically elite females, enhance the proportion of oocytes that become transferrable embryos, improve the fraction of embryos that establish pregnancy after transfer, reduce pregnancy wastage after pregnancy diagnosis, and identify culture conditions to optimize postnatal phenotype.

In vitro produced (IVP) embryos show large metabolic variability induced by breed, culture conditions, embryonic stage and sex and gamete donors. We hypothesized that the birth potential could be accurately predicted by UHPLC-MS/MS in culture medium (CM) with the discrimination of factors inducing metabolic variation. Day-6 embryos were developed in single CM (modified synthetic oviduct fluid) for 24 h and transferred to recipients as fresh (28 ETs) or frozen/thawed (58 ETs) Day-7 blastocysts. Variability was induced with seven bulls, slaughterhouse oocyte donors, culture conditions (serum + Bovine Serum Albumin [BSA] or BSA alone) prior to single culture embryonic stage records (Day-6: morula, early blastocyst, blastocyst; Day-7: expanding blastocyst; fully expanded blastocysts) and cryopreservation. Retained metabolite signals (6111) were analyzed as a function of pregnancy at Day-40, Day-62 and birth in a combinatorial block study with all fixed factors. We identified 34 accumulated metabolites through 511 blocks, 198 for birth, 166 for Day-62 and 147 for Day-40. The relative abundance of metabolites was higher within blocks from non-pregnant (460) than from pregnant (51) embryos. Taxonomy classified lipids (12 fatty acids and derivatives; 224 blocks), amino acids (12) and derivatives (3) (186 blocks), benzenoids (4; 58 blocks), tri-carboxylic acids (2; 41 blocks) and 5-Hydroxy-l-tryptophan (2 blocks). Some metabolites were effective as single biomarkers in 95 blocks (Receiver Operating Characteristic - Area Under the Curve [ROC-AUC]: 0.700-1.000). In contrast, more accurate predictions within the largest data sets were obtained with combinations of 2, 3 and 4 single metabolites in 206 blocks (ROC-AUC = 0.800-1.000). Pregnancy-prone embryos consumed more amino acids and citric acid, and depleted less lipids and cis-aconitic acid. Big metabolic differences between embryos support efficient pregnancy and birth prediction when analyzed in discriminant conditions.

Implantation and placentation are critical steps for successful pregnancy. The pig has a non-invasive placenta and the uterine luminal epithelium is intact throughout pregnancy. To better understand the regulation mechanisms in functions of endometrium at three certain gestational stages that are critical for embryo/fetal loss in pigs, we characterized microRNA (miRNA) expression profiles in the endometrium on days 15 (implantation period), 26 (placentation period) and 50 (mid-gestation period) of gestation. The differentially expressed miRNAs across gestational days were detected and of which, 65 miRNAs were grouped into 4 distinct categories according to the similarities in their temporal expression patterns: (1) categories A and B contain majority of miRNAs (51 miRNAs, such as the miR-181 family) that were down- or up-regulated between gestational days 15 and 26, respectively; (2) categories C and D (14 miRNAs) consist miRNAs that were down- or up-regulated between gestational days 26 and 50, respectively. The expression patterns represented by eleven miRNAs were validated by qPCR. The majority of miRNAs were in categories A and B, suggesting that these miRNAs were involved in regulation of embryo implantation and placentation. The pathway analysis revealed that the predicted targets were involved in several pathways, such as focal adhesion, cell proliferation and tissue remolding. Furthermore, we identified that genes well-known to affect embryo implantation in pigs, namely SPP1, ITGB3 and ESR1, contain the miR-181a or miR-181c binding sites using the luciferase reporter system. The present study revealed distinctive miRNA expression patterns in the porcine endometrium during the implantation, placentation or mid-gestation periods. Additionally, our results suggested that miR-181a and miR-181c likely play important roles in the regulation of genes and pathways that are known to be involved in embryo implantation and placentation in pigs.

During vesicular transport between the endoplasmic reticulum and the Golgi, members of the TMED/p24 protein family form hetero-oligomeric complexes that facilitate protein-cargo recognition as well as vesicle budding. In addition, they regulate each other's level of expression. Despite analyses of TMED/p24 protein distribution in mammalian cells, yeast, and C. elegans, little is known about the role of this family in vertebrate embryogenesis. We report the presence of a single point mutation in Tmed2/p24beta(1) in a mutant mouse line, 99J, identified in an ENU mutagenesis screen for recessive developmental abnormalities. This mutation does not affect Tmed2/p24beta(1) mRNA levels but results in loss of TMED2/p24beta(1) protein. Prior to death at mid-gestation, 99J homozygous mutant embryos exhibit developmental delay, abnormal rostral-caudal elongation, randomized heart looping, and absence of the labyrinth layer of the placenta. We find that Tmed2/p24beta(1) is normally expressed in tissues showing morphological defects in 99J mutant embryos and that these affected tissues lack the TMED2/p24beta(1) oligomerization partners, TMED7/p24gamma(3) and TMED10/p24delta(1). Our data reveal a requirement for TMED2/p24beta(1) protein in the morphogenesis of the mouse embryo and placenta.

In the early Drosophila melanogaster embryo, the gene regulatory network controlled by Dpp signaling is involved in the subdivision of dorsal ectoderm into the presumptive dorsal epidermis and amnioserosa. In this work, we aimed to identify new Dpp downstream targets involved in dorsal ectoderm patterning. We used oligonucleotide D. melanogaster microarrays to identify the set of genes that are differential expressed between wild type embryos and embryos that overexpress Dpp (nos-Gal4>UAS-dpp) during early stages of embryo development. By using this approach, we identified 358 genes whose relative abundance significantly increased in response to Dpp overexpression. Among them, we found the entire set of known Dpp target genes that function in dorsal ectoderm patterning (zen, doc, hnt, pnr, ush, tup, and others) in addition to several up-regulated genes of unknown functions. Spatial expression pattern of up-regulated genes in response to Dpp overexpression as well as their opposing transcriptional responses to Dpp loss- and gain-of-function indicated that they are new candidate target genes of Dpp signaling pathway. We further analyse one of the candidate genes, CG13653, which is expressed at the dorsal-most cells of the embryo during a restricted period of time. CG13653 orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa. We characterized the enhancer region of CG13653 and revealed that CG13653 is directly regulated by Dpp signaling pathway.

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear if these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear whether these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.

The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements in the trophectoderm cell lineage. Slightly later, expression in the visceral endoderm promotes anterior visceral endoderm formation and anterior-posterior axis specification. Early induction in the epiblast beginning at day 6 is necessary for nascent mesoderm to undergo epithelial to mesenchymal transition (EMT). Eomes acts in a temporally and spatially restricted manner to sequentially specify the yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, and axial mesoderm progenitors during gastrulation. Little is known about the underlying molecular mechanisms governing Eomes actions during the formation of these distinct progenitor cell populations. Here, we introduced a degron-tag and mCherry reporter sequence into the Eomes locus. Our experiments analyzing homozygously tagged embryonic stem cells and embryos demonstrate that the degron-tagged Eomes protein is fully functional. dTAG (degradation fusion tag) treatment in vitro results in rapid protein degradation and recapitulates the Eomes-null phenotype. However in utero administration of dTAG resulted in variable and lineage-specific degradation, likely reflecting diverse cell type-specific Eomes expression dynamics. Finally, we demonstrate that Eomes protein rapidly recovers following dTAG wash-out in vitro. The ability to temporally manipulate Eomes protein expression in combination with cell marking by the mCherry-reporter offers a powerful tool for dissecting Eomes-dependent functional roles in these diverse cell types in the early embryo.