A molecular mechanism of symmetry breaking in the early chick embryo

The first obvious sign of bilateral symmetry in mammalian and avian embryos is the appearance of the primitive streak in the future posterior region of a radially symmetric disc. The primitive streak marks the midline of the future embryo. The mechanisms responsible for positioning the primitive streak remain largely unknown. Here we combine experimental embryology and mathematical modelling to analyse the role of the TGFβ-related molecules BMP4 and Vg1/GDF1 in positioning the primitive streak. Bmp4 and Vg1 are first expressed throughout the embryo, and then become localised to the future anterior and posterior regions of the embryo, where they will, respectively, inhibit or induce formation of the primitive streak. We propose a model based on paracrine signalling to account for the separation of the two domains starting from a homogeneous array of cells, and thus for the topological transformation of a radially symmetric disc to a bilaterally symmetric embryo

Polyethylene bio-degradation by caterpillars of the wax moth Galleria mellonella

Plastics are synthetic polymers derived from fossil oil and largely resistant to biodegradation. Polyethylene (PE) and polypropylene (PP) represent ∼92% of total plastic production. PE is largely utilized in packaging, representing ∼40% of total demand for plastic products (www.plasticseurope.org) with over a trillion plastic bags used every year [1]. Plastic production has increased exponentially in the past 50 years (Figure S1A in Supplemental Information, published with this article online). In the 27 EU countries plus Norway and Switzerland up to 38% of plastic is discarded in landfills, with the rest utilized for recycling (26%) and energy recovery (36%) via combustion (www.plasticseurope.org), carrying a heavy environmental impact. Therefore, new solutions for plastic degradation are urgently needed. We report the fast bio-degradation of PE by larvae of the wax moth Galleria mellonella, producing ethylene glycol.F.B. is a Ramon y Cajal Fellow at the IBBTEC, in Santander, Spain. We thank Dr. Simone Ruggeri and Dr. Jenny Zhang for technical help. This work was funded by the Leverhulme foundation. P.B. and F.B. are co-founders of the company Baky.ltd. The authors declare no competing interest

Wax worm saliva and the enzymes therein are the key to polyethylene degradation by Galleria mellonella

Plastic degradation by biological systems with re-utilization of the by-products could be a future solution to the global threat of plastic waste accumulation. Here, we report that the saliva of Galleria mellonella larvae (wax worms) is capable of oxidizing and depolymerizing polyethylene (PE), one of the most produced and sturdy polyolefin-derived plastics. This effect is achieved after a few hours’ exposure at room temperature under physiological conditions (neutral pH). The wax worm saliva can overcome the bottleneck step in PE biodegradation, namely the initial oxidation step. Within the saliva, we identify two enzymes, belonging to the phenol oxidase family, that can reproduce the same effect. To the best of our knowledge, these enzymes are the first animal enzymes with this capability, opening the way to potential solutions for plastic waste management through bio-recycling/up-cycling

Plastic degradation by insect hexamerins: Near-atomic resolution structures of the polyethylene-degrading proteins from the wax worm saliva

14 p.-8 fig.Plastic waste management is a pressing ecological, social, and economic challenge. The saliva of the lepidopteran Galleria mellonella larvae is capable of oxidizing and depolymerizing polyethylene in hours at room temperature. Here, we analyze by cryo–electron microscopy (cryo-EM) G. mellonella’s saliva directly from the native source. The three-dimensional reconstructions reveal that the buccal secretion is mainly composed of four hexamerins belonging to the hemocyanin/phenoloxidase family, renamed Demetra, Cibeles, Ceres, and a previously unidentified factor termed Cora. Functional assays show that this factor, as its counterparts Demetra and Ceres, is also able to oxidize and degrade polyethylene. The cryo-EM data and the x-ray analysis from purified fractions show that they self-assemble primarily into three macromolecular complexes with striking structural differences that likely modulate their activity. Overall, these results establish the ground to further explore the hexamerins’ functionalities, their role in vivo, and their eventual biotechnological application.This work was funded by Roechling Stiftung to F.B., Consejo Superior de Investigaciones Cientificas (CSIC) to F.B., Ministerio de Ciencia e Innovación (grants PID2019-111215RB-100 and PDC2022-133955-I00) to T.T., Ministerio de Ciencia e Innovación (grant PID2019-111215RB-100) to T.T., the Generalitat de Catalunya (2017 SGR 1192) to M.S., the Spanish Ministry of Science, Innovation and Universities MCIN/AEI/10.13039/501100011033 ERDF “A way to make Europe” PID2021-129038NB-I00 to M.S., and MCIN/AEI/10.13039/501100011033 (grant PID2020-120275GB-I00) to E.A.-P.Peer reviewe

Wax worm saliva and the enzymes therein are the key to polyethylene degradation by Galleria mellonella

11 p.-6 fig.Plastic degradation by biological systems with re-utilization of the by-products can be the future solution to the global threat of plastic waste accumulation. We report that the saliva of Galleria mellonella larvae (wax worms) is capable of oxidizing and depolymerizing polyethylene (PE), one of the most produced and sturdy polyolefin-derived plastics. This effect is achieved after a few hours’ exposure at room temperature and physiological conditions (neutral pH). The wax worm saliva can indeed overcome the bottleneck step in PE biodegradation, that is the initial oxidation step. Within the saliva, we identified two enzymes that can reproduce the same effect. This is the first report of enzymes with this capability, opening up the way to new ground-breaking solutions for plastic waste management through bio-recycling/up-cycling.Roechling Stiftung to FB Consejo Superior de Investigaciones Científicas (CSIC) to FB NATO Science for Peace and Security Programme (Grant SPS G5536) to TT Junta de Castilla y León, Consejería de Educación y Cultura y Fondo Social Europeo (Grant BU263P18) to TT Ministerio de Ciencia e Innovación (Grant PID2019-111215RB-100) to TT The Generalitat de Catalunya (2017 SGR 1192) to MS Ministerio de Ciencia e Innovación (Grant BFU2017-89143-P) to EA-PN

The endogenous retrovirus ENS-1 provides active binding sites for transcription factors in embryonic stem cells that specify extra embryonic tissue

<p>Abstract</p> <p>Background</p> <p>Long terminal repeats (LTR) from endogenous retroviruses (ERV) are source of binding sites for transcription factors which affect the host regulatory networks in different cell types, including pluripotent cells. The embryonic epiblast is made of pluripotent cells that are subjected to opposite transcriptional regulatory networks to give rise to distinct embryonic and extraembryonic lineages. To assess the transcriptional contribution of ERV to early developmental processes, we have characterized <it>in vitro </it>and <it>in vivo </it>the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells.</p> <p>Results</p> <p>We show that <it>Ens-1 </it>LTR activity is controlled by two transcriptional pathways that drive pluripotent cells to alternative developmental fates. Indeed, both Nanog that maintains pluripotency and Gata4 that induces differentiation toward extraembryonic endoderm independently activate the LTR. Ets coactivators are required to support Gata factors' activity thus preventing inappropriate activation before epigenetic silencing occurs during differentiation. Consistent with their expression patterns during chick embryonic development, Gata4, Nanog and Ets1 are recruited on the LTR in embryonic stem cells; in the epiblast the complementary expression of Nanog and Gata/Ets correlates with the <it>Ens-1 </it>gene expression pattern; and Ens-1 transcripts are also detected in the hypoblast, an extraembryonic tissue expressing Gata4 and Ets2, but not Nanog. Accordingly, over expression of Gata4 in embryos induces an ectopic expression of <it>Ens-1</it>.</p> <p>Conclusion</p> <p>Our results show that <it>Ens-1 </it>LTR have co-opted conditions required for the emergence of extraembryonic tissues from pluripotent epiblasts cells. By providing pluripotent cells with intact binding sites for Gata, Nanog, or both, <it>Ens-1 </it>LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.</p

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way

A Comparative Analysis of Extra-Embryonic Endoderm Cell Lines

Prior to gastrulation in the mouse, all endodermal cells arise from the primitive endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives are generally referred to as extra-embryonic endoderm (ExEn) because the majority of these cells contribute to extra-embryonic lineages encompassing the visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the definitive endoderm (DE) forms by ingression of cells from the epiblast. The DE comprises most of the cells of the gut and its accessory organs. Despite their different origins and fates, there is a surprising amount of overlap in marker expression between the ExEn and DE, making it difficult to distinguish between these cell types by marker analysis. This is significant for two main reasons. First, because endodermal organs, such as the liver and pancreas, play important physiological roles in adult animals, much experimental effort has been directed in recent years toward the establishment of protocols for the efficient derivation of endodermal cell types in vitro. Conversely, factors secreted by the VE play pivotal roles that cannot be attributed to the DE in early axis formation, heart formation and the patterning of the anterior nervous system. Thus, efforts in both of these areas have been hampered by a lack of markers that clearly distinguish between ExEn and DE. To further understand the ExEn we have undertaken a comparative analysis of three ExEn-like cell lines (END2, PYS2 and XEN). PYS2 cells are derived from embryonal carcinomas (EC) of 129 strain mice and have been characterized as parietal endoderm-like [1], END2 cells are derived from P19 ECs and described as visceral endoderm-like, while XEN cells are derived from blastocyst stage embryos and are described as primitive endoderm-like. Our analysis suggests that none of these cell lines represent a bona fide single in vivo lineage. Both PYS2 and XEN cells represent mixed populations expressing markers for several ExEn lineages. Conversely END2 cells, which were previously characterized as VE-like, fail to express many markers that are widely expressed in the VE, but instead express markers for only a subset of the VE, the anterior visceral endoderm. In addition END2 cells also express markers for the PE. We extended these observations with microarray analysis which was used to probe and refine previously published data sets of genes proposed to distinguish between DE and VE. Finally, genome-wide pathway analysis revealed that SMAD-independent TGFbeta signaling through a TAK1/p38/JNK or TAK1/NLK pathway may represent one mode of intracellular signaling shared by all three of these lines, and suggests that factors downstream of these pathways may mediate some functions of the ExEn. These studies represent the first step in the development of XEN cells as a powerful molecular genetic tool to study the endodermal signals that mediate the important developmental functions of the extra-embryonic endoderm. Our data refine our current knowledge of markers that distinguish various subtypes of endoderm. In addition, pathway analysis suggests that the ExEn may mediate some of its functions through a non-classical MAP Kinase signaling pathway downstream of TAK1

Distinct Steps of Neural Induction Revealed by Asterix, Obelix and TrkC, Genes Induced by Different Signals from the Organizer

The amniote organizer (Hensen's node) can induce a complete nervous system when grafted into a peripheral region of a host embryo. Although BMP inhibition has been implicated in neural induction, non-neural cells cannot respond to BMP antagonists unless previously exposed to a node graft for at least 5 hours before BMP inhibitors. To define signals and responses during the first 5 hours of node signals, a differential screen was conducted. Here we describe three early response genes: two of them, Asterix and Obelix, encode previously undescribed proteins of unknown function but Obelix appears to be a nuclear RNA-binding protein. The third is TrkC, a neurotrophin receptor. All three genes are induced by a node graft within 4–5 hours but they differ in the extent to which they are inducible by FGF: FGF is both necessary and sufficient to induce Asterix, sufficient but not necessary to induce Obelix and neither sufficient nor necessary for induction of TrkC. These genes are also not induced by retinoic acid, Noggin, Chordin, Dkk1, Cerberus, HGF/SF, Somatostatin or ionomycin-mediated Calcium entry. Comparison of the expression and regulation of these genes with other early neural markers reveals three distinct “epochs”, or temporal waves, of gene expression accompanying neural induction by a grafted organizer, which are mirrored by specific stages of normal neural plate development. The results are consistent with neural induction being a cascade of responses elicited by different signals, culminating in the formation of a patterned nervous system