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Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that bone morphogenetic protein (BMP) signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hr after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification., Competing Interests: NS, JW, AC, LJ, LT, XM, SR, JS, TG, CL, KT No competing interests declared, (© 2023, Sekulovski et al.)

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos 1-8 . Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells 9-17 . Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis., (© 2023. The Author(s).)

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system 1,2 . Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons 3-5 . Stem cell models of human embryo have emerged to help unlock the mysteries of this stage 6-16 . Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling., (© 2023. The Author(s).)

Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development., (© 2021. The Author(s).)

Recent advances in synthetic human embryology has provided a previously inexistent molecular portrait of human development. Models of synthetic human embryonic tissues capitalize on the self-organizing capabilities of human embryonic stem cells when they are cultured on biomimetic conditions that simulate in vivo human development. In this Review, we discuss these models and how they have shed light on the early stages of human development including amniotic sac development, gastrulation and neurulation. We discuss the mechanisms underlying the molecular logic of embryonic tissue self-organization that have been dissected using synthetic models of human embryology and explore future challenges in the field. Geared with technological advances in bioengineering, high resolution gene expression and imaging tools, these models are set to transform our understanding of the mechanistic basis of embryonic tissue self-organization during human development and how they may go awry in disease., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mammalian embryogenesis is a paradigm of regulative development as mouse embryos show plasticity in the regulation of cell fate, cell number, and tissue morphogenesis. However, the mechanisms behind embryo plasticity remain largely unknown. Here, we determine how mouse embryos respond to an increase in cell numbers to regulate the timing and mechanism of embryonic morphogenesis, leading to the formation of the pro-amniotic cavity. Using embryos and embryonic stem cell aggregates of different size, we show that while pro-amniotic cavity formation in normal-sized embryos is achieved through basement membrane-induced polarization and exocytosis, cavity formation of increased-size embryos is delayed and achieved through apoptosis of cells that lack contact with the basement membrane. Importantly, blocking apoptosis, both genetically and pharmacologically, alters pro-amniotic cavity formation but does not affect size regulation in enlarged embryos. We conclude that the regulation of embryonic size and morphogenesis, albeit concomitant, have distinct molecular underpinnings., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation 1 , exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect 2,3 . Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud 3 . In the neuromesodermal precursors of the tail bud 4 , WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate 3,5 . How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells 6 . Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic β-catenin acetylation downstream of WNT signalling. As acetylated β-catenin promotes mesodermal rather than neural fate 7 , this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.

Objectives: To evaluate the incidence and types of chromosomal abnormalities detected in twins with structural anomalies and compare their distribution according to chorionicity and amnionicity and by structural-anomaly type. The added value of chromosomal microarray analysis (CMA) over conventional karyotyping in twins was also estimated., Methods: This was a single-center, retrospective analysis of 534 twin pregnancies seen over an 11-year period, in which one or both fetuses were diagnosed with congenital structural anomalies on ultrasound. The ultrasound findings and invasive prenatal diagnostic results were reviewed. Twin pregnancies were categorized as monochorionic monoamniotic (MCMA), monochorionic diamniotic (MCDA) or dichorionic diamniotic (DCDA). Chromosomal abnormalities detected by G-banding karyotyping and/or CMA were analyzed by chorionicity and amnionicity and by structural-anomaly type., Results: The 534 twin pairs analyzed comprised 25 pairs of MCMA, 112 pairs of MCDA and 397 pairs of DCDA twins. Of the 549 fetuses affected by structural anomalies, 432 (78.7%) underwent invasive prenatal testing and cytogenetic results were obtained. The incidence of overall chromosomal abnormalities in the DCDA fetuses (25.4%) was higher than that in the MCMA (3.7%) and MCDA (15.3%) fetuses. The incidence of aneuploidy was significantly higher in the DCDA group (22.8%) than in the MCMA (0.0%) and MCDA (12.4%) groups. The incidence of chromosomal abnormalities detected in fetuses, with anomalies of the cardiovascular, faciocervical, musculoskeletal, genitourinary and gastrointestinal systems, was higher in the DCDA group than in the MCDA group. In both the DCDA and MCDA groups, hydrops fetalis was associated with the highest incidence of chromosomal abnormality; of these fetuses, 67.6% had Turner syndrome (45,X). Pathogenic copy-number variations (CNVs) undetectable by karyotyping were identified by CMA in five (2.0%; 95% CI, 0.3-3.7%) DCDA fetuses. No pathogenic CNVs were found in MCMA and MCDA twins., Conclusions: Dichorionic twins with structural anomalies have a higher risk of chromosomal abnormalities, especially aneuploidies, than do monochorionic twins. The incremental diagnostic yield of CMA over karyotyping seems to be lower (2.0%) in twins than that reported in singleton pregnancy. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd., (Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.)

Context: Prenatal exposure to bisphenol A (BPA) is linked to obesity and diabetes but the molecular mechanisms driving these phenomena are not known. Alterations in deoxyribonucleic acid (DNA) methylation in amniocytes exposed to BPA in utero represent a potential mechanism leading to metabolic dysfunction later in life., Objective: To profile changes in genome-wide DNA methylation and expression in second trimester human amniocytes exposed to BPA in utero., Design: A nested case-control study was performed in amniocytes matched for offspring sex, maternal race/ethnicity, maternal age, gestational age at amniocentesis, and gestational age at birth. Cases had amniotic fluid BPA measuring 0.251 to 23.74 ng/mL. Sex-specific genome-wide DNA methylation analysis and RNA-sequencing (RNA-seq) were performed to determine differentially methylated regions (DMRs) and gene expression changes associated with BPA exposure. Ingenuity pathway analysis was performed to identify biologically relevant pathways enriched after BPA exposure. In silico Hi-C analysis identified potential chromatin interactions with DMRs., Results: There were 101 genes with altered expression in male amniocytes exposed to BPA (q < 0.05) in utero, with enrichment of pathways critical to hepatic dysfunction, collagen signaling and adipogenesis. Thirty-six DMRs were identified in male BPA-exposed amniocytes and 14 in female amniocyte analysis (q < 0.05). Hi-C analysis identified interactions between DMRs and 24 genes with expression changes in male amniocytes and 12 in female amniocytes (P < 0.05)., Conclusion: In a unique repository of human amniocytes exposed to BPA in utero, sex-specific analyses identified gene expression changes in pathways associated with metabolic disease and novel DMRs with potential distal regulatory functions., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms 1,2 that do not recapitulate the in vivo conditions 3-5 . Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior-posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning 1 . Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence 2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.

Extraembryonic tissues contribute to animal development, which often entails spreading over embryo or yolk. Apart from changes in cell shape, the requirements for this tissue spreading are not well understood. Here, we analyze spreading of the extraembryonic serosa in the scuttle fly Megaselia abdita . The serosa forms from a columnar blastoderm anlage, becomes a squamous epithelium, and eventually spreads over the embryo proper. We describe the dynamics of this process in long-term, whole-embryo time-lapse recordings, demonstrating that free serosa spreading is preceded by a prolonged pause in tissue expansion. Closer examination of this pause reveals mechanical coupling to the underlying yolk sac, which is later released. We find mechanical coupling prolonged and serosa spreading impaired after knockdown of M. abdita Matrix metalloprotease 1 . We conclude that tissue-tissue interactions provide a critical functional element to constrain spreading epithelia., Competing Interests: FC, EG, VN, PG, DK, MW, LS, LH, SL No competing interests declared, (© 2018, Caroti et al.)

Upon gastrulation, the mammalian conceptus transforms rapidly from a simple bilayer into a multilayered embryo enveloped by its extra-embryonic membranes. Impaired development of the amnion, the innermost membrane, causes major malformations. To clarify the origin of the mouse amnion, we used single-cell labelling and clonal analysis. We identified four clone types with distinct clonal growth patterns in amniotic ectoderm. Two main types have progenitors in extreme proximal-anterior epiblast. Early descendants initiate and expand amniotic ectoderm posteriorly, while descendants of cells remaining anteriorly later expand amniotic ectoderm from its anterior side. Amniogenesis is abnormal in embryos deficient in the bone morphogenetic protein (BMP) signalling effector SMAD5, with delayed closure of the proamniotic canal, and aberrant amnion and folding morphogenesis. Transcriptomics of individual Smad5 mutant amnions isolated before visible malformations and tetraploid chimera analysis revealed two amnion defect sets. We attribute them to impairment of progenitors of the two main cell populations in amniotic ectoderm and to compromised cuboidal-to-squamous transition of anterior amniotic ectoderm. In both cases, SMAD5 is crucial for expanding amniotic ectoderm rapidly into a stretchable squamous sheet to accommodate exocoelom expansion, axial growth and folding morphogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Development of the asymmetric amniotic sac-with the embryonic disc and amniotic ectoderm occupying opposite poles-is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.Early in human embryonic development, it is unclear how amniotic sac formation is regulated. Here, the authors use a human pluripotent stem cell-based model, termed the post-implantation amniotic sac embryoid, to recapitulate early embryogenic events of human amniotic sac development.

Mammalian embryos transiently exhibit aerobic glycolysis (Warburg effect), a metabolic adaptation also observed in cancer cells. The role of this particular type of metabolism during vertebrate organogenesis is currently unknown. Here, we provide evidence for spatiotemporal regulation of glycolysis in the posterior region of mouse and chicken embryos. We show that a posterior glycolytic gradient is established in response to graded transcription of glycolytic enzymes downstream of fibroblast growth factor (FGF) signaling. We demonstrate that glycolysis controls posterior elongation of the embryonic axis by regulating cell motility in the presomitic mesoderm and by controlling specification of the paraxial mesoderm fate in the tail bud. Our results suggest that glycolysis in the tail bud coordinates Wnt and FGF signaling to promote elongation of the embryonic axis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Transplanting vascular endothelial cells (ECs) to support metabolism and express regenerative paracrine factors is a strategy to treat vasculopathies and to promote tissue regeneration. However, transplantation strategies have been challenging to develop, because ECs are difficult to culture and little is known about how to direct them to stably integrate into vasculature. Here we show that only amniotic cells could convert to cells that maintain EC gene expression. Even so, these converted cells perform sub-optimally in transplantation studies. Constitutive Akt signalling increases expression of EC morphogenesis genes, including Sox17, shifts the genomic targeting of Fli1 to favour nearby Sox consensus sites and enhances the vascular function of converted cells. Enforced expression of Sox17 increases expression of morphogenesis genes and promotes integration of transplanted converted cells into injured vessels. Thus, Ets transcription factors specify non-vascular, amniotic cells to EC-like cells, whereas Sox17 expression is required to confer EC function., Competing Interests: M.G. is a senior scientist at Angiocrine Bioscience. The remaining other authors declare no competing financial interests.

The detection of specific RNA molecules in embryonic tissues has wide research applications including studying gene expression dynamics in brain development and evolution. Recent advances in sequencing technologies have introduced new animal models to explore the molecular principles underlying the assembly and diversification of brain circuits between different amniote species. Here, we provide a step-by-step protocol for a versatile in situ hybridization method that is immediately applicable to a range of amniote embryos including zebra finch and Madagascar ground gecko, two new model organisms that have rapidly emerged for comparative brain studies over recent years. The sensitive detection of transcripts from low to high abundance expression range using the same platform enables direct comparison of gene of interest among different amniotes, providing high-resolution spatiotemporal information of gene expression to dissect the molecular principles underlying brain evolution.

Introduction: Although mortality has decreased for fetuses with lower urinary tract obstruction treated with vesicoamniotic shunt (VAS) placement, survivors remain at risk for long-term renal impairment. We tested the association of fetal serum β2-microglobulin (fsβ2M) with postnatal renal function in these patients, hypothesizing that fsβ2M may predict such renal impairment., Materials and Methods: fsβ2M was obtained in patients undergoing VAS placement. The primary outcome was renal function at 3-12 months of life, as assessed by a pediatric nephrologist using medical records. Patients were divided into two groups: (1) 'stable renal function' - probable stable long-term renal function and reasonable growth - and (2) 'loss of renal function' - early loss of renal function and failure to thrive., Results: Nineteen patients with preoperative fsβ2M received a VAS. Of the 14 survivors, those with fsβ2M ≤5.6 mg/l tended to have stable renal function compared to those with fsβ2M >5.6 mg/l [5/6 (83.3%) vs. 2/8 (25.0%), OR = 15.00, 95% CI 0.70-709.89; p = 0.1026]. Eight of 9 patients followed for >12 months of age had outcomes consistent with the initial renal assessments., Discussion: Patients with initial fsβ2M >5.6 mg/l and treated with VAS tended to have poor renal outcomes., (© 2016 S. Karger AG, Basel.)

Dorsal closure is an essential stage of Drosophila embryogenesis and is a powerful model system for morphogenesis, wound healing, and tissue biomechanics. During closure, two flanks of lateral epidermis close an eye-shaped dorsal opening that is filled with amnioserosa. The two flanks of lateral epidermis are zipped together at each canthus ("corner" of the eye). Actomyosin-rich purse strings are localized at each of the two leading edges of lateral epidermis ("lids" of the eye). Here we report that each purse string indents the dorsal surface at each leading edge. The amnioserosa tissue bulges outward during the early-to-mid stages of closure to form a remarkably smooth, asymmetric dome indicative of an isotropic and uniform surface tension. Internal pressure of the embryo and tissue elastic properties help to shape the dorsal surface., (© 2016 Lu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

We present a detailed study of the formation of the amniotic sac in the avian embryo, and a comparison with the crocodile amniotic sac. We show that the amniotic sac forms at a circular line of stiffness contrast, separating rings of cell domains. Cells align at this boundary, and this in turn orients and concentrates the tension forces. The tissue fold which forms the amniotic sac is locked exactly along this line due to the colocalization of the stiffness contrast and of the tensile force. In addition, the tensile force plays a regenerative role when the amniotic sac is cut. The fold forming the ventral side of the embryo displays the same characteristics. This work shows that amniote embryogenesis consists of a cascade of buckling events taking place at the boundaries between regions of differing mechanical properties. Hence, amniote embryogenesis relies on a simple and robust biomechanical scheme used repeatedly, and selected ancestrally.

Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSR) in 49 potential target proteins. Nearly half the POFUT2 targets belong to the A Disintegrin and Metalloprotease with ThromboSpondin type-1 motifs (ADAMTS) or ADAMTS-like family of proteins. Both the mouse Pofut2 RST434 gene trap allele and the Adamts9 knockout were reported to result in early embryonic lethality, suggesting that defects in Pofut2 mutant embryos could result from loss of O-fucosylation on ADAMTS9. To address this question, we compared the Pofut2 and Adamts9 knockout phenotypes and used Cre-mediated deletion of Pofut2 and Adamts9 to dissect the tissue-specific role of O-fucosylated ADAMTS9 during gastrulation. Disruption of Pofut2 using the knockout (LoxP) or gene trap (RST434) allele, as well as deletion of Adamts9, resulted in disorganized epithelia (epiblast, extraembryonic ectoderm, and visceral endoderm) and blocked mesoderm formation during gastrulation. The similarity between Pofut2 and Adamts9 mutants suggested that disruption of ADAMTS9 function could be responsible for the gastrulation defects observed in Pofut2 mutants. Consistent with this prediction, CRISPR/Cas9 knockout of POFUT2 in HEK293T cells blocked secretion of ADAMTS9. We determined that Adamts9 was dynamically expressed during mouse gastrulation by trophoblast giant cells, parietal endoderm, the most proximal visceral endoderm adjacent to the ectoplacental cone, extraembryonic mesoderm, and anterior primitive streak. Conditional deletion of either Pofut2 or Adamts9 in the epiblast rescues the gastrulation defects, and identified a new role for O-fucosylated ADAMTS9 during morphogenesis of the amnion and axial mesendoderm. Combined, these results suggested that loss of ADAMTS9 function in the extra embryonic tissue is responsible for gastrulation defects in the Pofut2 knockout. We hypothesize that loss of ADAMTS9 function in the most proximal visceral endoderm leads to slippage of the visceral endoderm and altered characteristics of the extraembryonic ectoderm. Consequently, loss of input from the extraembryonic ectoderm and/or compression of the epiblast by Reichert's membrane blocks gastrulation. In the future, the Pofut2 and Adamts9 knockouts will be valuable tools for understanding how local changes in the properties of the extracellular matrix influence the organization of tissues during mammalian development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy.

Despite being a short-lived, extraembryonic tissue, the amnioserosa plays critical roles in the major morphogenetic events of Drosophila embryogenesis. These roles involve both cellular mechanics and biochemical signaling. Its best-known role is in dorsal closure-well studied by both developmental biologists and biophysicists-but the amnioserosa is also important during earlier developmental stages. Here, we provide an overview of amnioserosa specification and its role in several key developmental stages: germ band extension, germ band retraction, and dorsal closure. We also compare embryonic development in Drosophila and its relative Megaselia to highlight how the amnioserosa and its roles have evolved. Placed in context, the amnioserosa provides a fascinating example of how signaling, mechanics, and morphogen patterns govern cell-type specification and subsequent morphogenetic changes in cell shape, orientation, and movement. Developmental Dynamics 245:558-568, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The mechanisms responsible for twinning and disorders of twin gestations have been the subject of considerable interest by physicians and scientists, and cases of atypical twinning have called for a reexamination of the fundamental theories invoked to explain twin gestations. This article presents a review of the literature focusing on twinning and atypical twinning with an emphasis on the phenomena of chimeric twins, phenotypically discordant monozygotic twins, mirror-image twins, polar body twins, complete hydatidiform mole with a coexistent twin, vanishing twins, fetus papyraceus, fetus in fetu, superfetation, and superfecundation. The traditional models attributing monozygotic twinning to a fission event, and more recent models describing monozygotic twinning as a fusion event, are critically reviewed. Ethical restrictions on scientific experimentation with human embryos and the rarity of cases of atypical twinning have limited opportunities to elucidate the exact mechanisms by which these phenomena occur. Refinements in the modeling of early embryonic development in twin pregnancies may have significant clinical implications. The article includes a series of figures to illustrate the phenomena described., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Most insect embryos develop with two distinct extraembryonic membranes, the serosa and the amnion. In the insect beetle Tribolium the early origin of the serosa within the anterior blastoderm is well established but the origin of the amnion is still debated. It is not known whether this tissue develops from a blastodermal precursor or originates de novo later from embryonic tissue during embryogenesis. We undertook an in-depth analysis of the spatio-temporal expression pattern profile of important extraembryonic membrane marker genes with emphasis on early blastoderm development in Tribolium. The amnion marker iroquois (Tc-iro) was found co-expressed with the serosa marker zerknüllt1 (Tc-zen1) during early blastoderm formation in an anterior cap domain. This domain later resolved into two adjacent domains that likely represent the precursors of the serosa and the amnion. In addition, we found the hindsight ortholog in Tribolium (Tc-hnt) to be a serosa-specific marker. Surprisingly, decapentaplegic (Tc-dpp) expression was not seen as a symmetric cap domain but detected asymmetrically first along the DV- and later also along the AP-axis. Moreover, we found a previously undescribed domain of phosphorylated MAD (pMAD) protein in anterior ventral serosal cells. This is the first study showing that the anterior-lateral part of the amnion originates from the anterior blastoderm while the precursor of the dorsal amnion develops later de novo from a dorsal-posterior region within the differentiated blastoderm., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Placental type 3 iodothyronine deiodinase (D3) potentially protects the fetus from the elevated maternal thyroid hormones. Na(+)/I(-) symporter (NIS) is a plasma membrane glycoprotein, which mediates active iodide uptake. Our objectives were to establish the distribution of NIS and D3 gene expressions in the placenta and the amniotic membrane and to investigate the relationship between placental D3 and NIS gene expressions and maternal iodine, selenium, and thyroid hormone status. Thyroid hormones, urinary iodine concentration (UIC), and selenium levels were measured in 49 healthy term pregnant women. NIS and D3 gene expressions were studied with the total mRNA RT-PCR method in tissues from maternal placenta (n = 49), fetal placenta (n = 9), and amniotic membrane (n = 9). NIS and D3 gene expressions were shown in the fetal and maternal sides of the placenta and amniotic membrane. Mean blood selenium level was 66 ± 26.5 μg/l, and median UIC was 143 μg/l. We could not demonstrate any statistically significant relationship of spot UIC and blood selenium with NIS and D3 expression (p > 0.05). Positive correlations were found between NIS and thyroxine-binding globulin (TBG) (r = 0.3, p = 0.042) and between D3 and preoperative glucose levels (r = 0.4, p = 0.006). D3 and NIS genes are expressed in term placenta and amniotic membrane; thus, in addition to placenta, amniotic membrane contributes to regulation of maternofetal iodine and thyroid hormone transmission. Further studies are needed to clarify the relationship between maternal glucose levels and placental D3 expression and between TBG and placental NIS expression.

Studies on new arthropod models such as the beetle Tribolium castaneum are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, Tribolium embryos exhibit the short-germ type of development and become enveloped by extensive extra-embryonic membranes, the amnion and serosa. The genetic basis of these processes has been the focus of active research. Here, we complement genetic approaches with live fluorescence imaging of Tribolium embryos to make the link between gene function and morphogenetic cell behaviors during blastoderm formation and differentiation, germband condensation and elongation, and extra-embryonic development. We first show that transient labeling methods result in strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labeling the chromatin, membrane, cytoskeleton or combinations thereof. We then use co-injection of fluorescent markers with dsRNA for live imaging of embryos with disrupted caudal gene function caused by RNA interference. Using these approaches, we describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. We find that Tribolium germband condensation is effected by cell contraction and intercalation, with the latter being dependent on the anterior-posterior patterning system. We propose that germband condensation drives initiation of amnion folding, whereas expansion of the amniotic fold and closure of the amniotic cavity are likely driven by contraction of an actomyosin cable at the boundary between the amnion and serosa. Our methodology provides a comprehensive framework for testing quantitative models of patterning, growth and morphogenetic mechanisms in Tribolium and other arthropod species.

Inflammation of the uterine environment (commonly as a result of microbial colonisation of the fetal membranes, amniotic fluid and fetus) is strongly associated with preterm labour and birth. Both preterm birth and fetal inflammation are independently associated with elevated risks of subsequent short- and long-term respiratory, gastro-intestinal and neurological complications. Despite numerous clinical and experimental studies to investigate localised and systemic fetal inflammation following exposure to microbial agonists, there is minimal data to describe which fetal organ(s) drive systemic fetal inflammation. We used lipopolysaccharide (LPS) from E.coli in an instrumented ovine model of fetal inflammation and conducted a series of experiments to assess the systemic pro-inflammatory capacity of the three major fetal surfaces exposed to inflammatory mediators in pregnancy (the lung, gastro-intestinal tract and skin/amnion). Exposure of the fetal lung and fetal skin/amnion (but not gastro-intestinal tract) caused a significant acute systemic inflammatory response characterised by altered leucocytosis, neutrophilia, elevated plasma MCP-1 levels and inflammation of the fetal liver and spleen. These novel findings reveal differential fetal organ responses to pro-inflammatory stimulation and shed light on the pathogenesis of fetal systemic inflammation after exposure to chorioamnionitis.

Bone morphogenetic protein (BMP) signaling is an essential factor in dorsoventral patterning of animal embryos but how BMP signaling evolved with fundamental changes in dorsoventral tissue differentiation is unclear. Flies experienced an evolutionary reduction of extra-embryonic tissue types from two (amniotic and serosal tissue) to one (amnionserosal tissue). BMP-dependent amnioserosa specification has been studied in Drosophila melanogaster. However, the mechanisms of serosal and amniotic tissue specification in less diverged flies remain unknown. To better understand potential evolutionary links between BMP signaling and extra-embryonic tissue specification, we examined the activity profile and function of BMP signaling in serosa and amnion patterning of the scuttle fly Megaselia abdita (Phoridae) and compared the BMP activity profiles between M. abdita and D. melanogaster. In blastoderm embryos of both species, BMP activity peaked at the dorsal midline. However, at the beginning of gastrulation, peak BMP activity in M. abdita shifted towards prospective amnion tissue. This transition correlated with the first signs of amnion differentiation laterally adjacent to the serosa anlage. Marker-assisted analysis of six BMP signaling components (dpp, gbb, scw, tkv, sax, sog) by RNA interference revealed that both serosa and amnion specification of M. abdita are dependent on BMP activity. Conversely, BMP gain-of-function experiments caused sharpened expression boundaries of extra-embryonic target genes indicative of positive feedback. We propose that changes in the BMP activity profile at the beginning of gastrulation might have contributed to the reduction of extra-embryonic tissue types during the radiation of cyclorrhaphan flies.

During Drosophila embryogenesis the majority of the extra-embryonic epithelium known as the amnioserosa (AS) undergoes programmed cell death (PCD) following the completion of the morphogenetic process of dorsal closure. Approximately ten percent of AS cells, however, are eliminated during dorsal closure by extrusion from the epithelium. Using biosensors that report autophagy and caspase activity in vivo, we demonstrate that AS cell extrusion occurs in the context of elevated autophagy and caspase activation. Furthermore, we evaluate AS extrusion rates, autophagy, and caspase activation in embryos in which caspase activity or autophagy are altered by genetic manipulation. This includes using the GAL4/UAS system to drive expression of p35, reaper, dINR (ACT) and Atg1 in the AS; we also analyze embryos lacking both maternal and zygotic expression of Atg1. Based on our results we suggest that autophagy can promote, but is not required for, epithelial extrusion and caspase activation in the amnioserosa.

Background: Despite the detailed knowledge obtained over the last decade on the molecular regulation of gastrulation in amniotes, the process of amnion development has been poorly described and illustrated in mice, and conflicting descriptions exist. Understanding the morphogenesis and development not only of the early mouse embryo, but also of its extraembryonic tissues, is crucial for correctly interpreting fate-mapping data and mouse mutants with gastrulation defects. Moreover, the recent isolation from amnion of cells with stem cell features further argues for a better understanding of the process of amnion formation. Here, we revisit the highly dynamic process of amnion formation in the mouse. Amnion development starts early during gastrulation and is intimately related to the formation of the exocoelom and the expansion of the amniotic fold. The authoritative description involves the fusion of two amniotic folds, a big posterior and a smaller anterior fold. We challenged this 'two amniotic folds' model by performing detailed histomorphological analyses of dissected, staged embryos and 3D reconstructions using historical sections., Results: A posterior fold of extraembryonic ectoderm and associated epiblast is formed early during gastrulation by accumulation of extraembryonic mesoderm posterior to the primitive streak. Previously called the "posterior amniotic fold", we rename it the "amniochorionic fold" (ACF) because it forms both amnion and chorion. Exocoelom formation within the ACF seems not to involve apoptosis within the mesoderm. The ACF and exocoelom expand without disrupting the anterior junction of epiblast, extraembryonic ectoderm and visceral endoderm. No separate anterior fold is formed; its absence was confirmed in 3D reconstructions. Amnion and chorion closure is eccentric, close to the anterior margin of the egg cylinder: we name it the "anterior separation point"., Conclusions: Here, we reconcile previous descriptions of amnion formation and provide new nomenclature, as well as an animation, that clarify and emphasize the arrangement of the tissues that contribute to amnion development and the dynamics of the process. According to our data, the amnion and the chorion are formed by a single amniochorionic fold initiated posteriorly. Finally, we give an overview on mutant mouse models with impaired amnion development.

This study examined the process of chick amniogenesis to determine whether the actin cable mechanism operating during amnion wound repair is a recapitulation of developmental events. Staining of the developing amnion with fluorescein isothiocyanate-labeled phalloidin indicated the presence of an actin cable in the amniotic head fold, which persisted through to the closure of the amnion. Transmission electron microscopy of the developing amnion revealed linearly arranged actin microfilaments in the elongated cells at the leading edge of the amnion, adjacent to either side of a nodule of numerous cells at the point of midline fusion. A mesh of cytoplasmic actin filaments was seen dispersed throughout the accumulated cells of the nodule. Lamellapodia were absent suggesting that cell crawling is not involved in amniogenesis. Addition of an enzyme inhibitor of Rho, cell-permeable C3 transferase, to the surface of the developing amnion prior to closure appeared to inhibit amniogenesis at the early embryonic stages., (Copyright © 2011 Wiley-Liss, Inc.)

Purpose: To evaluate the efficacy of cultured cutaneous substitute (CCS) in accelerating the healing of combined radiation-skin wound injury (CRWI) in minipigs., Material and Methods: Autologous porcine bone marrow-derived mesenchymal stem cells (BMSC) and skin-derived keratinocytes (SK) were infected by recombinant retrovirus expressing human (h) platelet-derived growth factor-A (hPDGF-A). CCS was constructed by loading acellular human amniotic membrane (HAM) with normal porcine BMSC and SK (BMSC-/SK-CCS) or with hPDGF-A modified counterparts (BMSC+/SK+CCS). The expression of exogenous hPDGF-A in cells and CCS was assessed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The CCS or HAM were grafted to the dorsal CRWI sites (20 Gy local irradiation plus full-thickness skin removal, diameter = 40 mm) of minipigs. Wound healing rate and pathological changes were observed., Results: High levels of hPDGF-A expression were confirmed in gene-modified cells (3780 pg/ml), cultured CCS (506 pg/ml) and transplanted CCS (250 pg/ml). The transplantation of the BMSC+/SK+CCS resulted in a shorter healing time (16-18, days) (P < 0.05 vs. other groups). The healing rates ranked as BMSC+/SK+CCS > BMSC-/SK-CCS > HAM > wound control. Pathologically, there were better granulation formation and re-epithelialisation, and collagen deposition in BMSC+/SK+CCS-treated wound than those in other groups. The angiogenesis ability followed the same order as healing rate of different groups. At day 7, the area densities of vasculature in granulation tissue of group BMSC+/SK+CCS, BMSC-/SK-CCS, HAM, wound only were 15.4, 10.3, 6.0 and 5.7%, respectively, while the number densities of vasculature was 767, 691, 126 and 109 (number/mm(2)), respectively., Conclusions: Topical transplantation of hPDGF-A modified CCS may be applicable to the management of refractory wounds.

As extra-embryonic tissues, the amnion and serosa are not considered to contribute materially to the insect embryo, yet they must execute an array of morphogenetic movements before they are dispensable. In hemimetabolous insects, these movements have been known for over a century, but they have remained virtually unexamined. This study addresses late extraembryonic morphogenesis in the milkweed bug, Oncopeltus fasciatus. Cell shape changes and apoptosis profiles are used to characterize the membranes as they undergo a large repertoire of final reorganizational events that reposition the embryo (katatrepsis), and eliminate the membranes themselves in an ordered fashion (dorsal closure). A number of key features were identified. First, amnion-serosa "fusion" involves localized apoptosis in the amnion and the formation of a supracellular actin purse string at the amnion-serosa border. During katatrepsis, a 'focus' of serosal cells undergoes precocious columnarization and may serve as an anchor for contraction. Lastly, dorsal closure involves novel modifications of the amnion and embryonic flank that are without counterpart during the well-known process of dorsal closure in the fruit fly Drosophila melanogaster. These data also address the long-standing question of the final fate of the amnion: it undergoes apoptosis during dorsal closure and thus is likely to be solely extraembryonic., (Copyright 2010 Elsevier Inc. All rights reserved.)

A common characteristic of mammals is the development of extraembryonic supporting tissues and organs that are required for embryonic implantation, survival and development in utero. The amnion is the innermost extraembryonic membrane that eventually surrounds the fetus of amniotes, and contains the amniotic fluid. Next to its function in in utero development, the amnion has been shown to have an important potential for clinical applications. It is mainly used as a dressing to stimulate healing in skin and ocular wounds. Moreover, cells derived from the amniotic membrane and amniotic fluid have been reported to possess stem cell features, like pluripotent differentiation ability. Little is known about the early development of this membrane in humans. The mouse is a powerful genetic model organism that can be used to address the dynamics and the developmental origin of amnion and amnion-derived stem cells. Here, we discuss some fundamental differences in amnion development in the disc-shaped primate embryo and in the cup-shaped mouse embryo. We emphasize the consequences that this may have on the derivation of amniotic "stem" cells. After revision of the different isolation procedures of amniotic (fluid) derived "stem" cells from rodents, we reveal striking differences in the sources used to derive these cells across studies. The profound differences in the development of the extraembryonic membranes and cavities between primates and rodents may result in comparing cell types of different developmental origins, eventually leading to missinterpretations.

Objective: Numerous researchers have evaluated the insulin-like growth factors (IGF) influence on mammalian fetal development. Although IGF has been explored in the avian system, questions remain on the role of IGF in avian development. Therefore, the current study evaluated the mRNA abundance of IGF in the amnion and allantoic membranes and developing small intestine in the chicken, duck, and turkey during the incubation and post-hatch period., Design: Broiler, duck, and turkey eggs were incubated with small intestinal, allantoic, and amniotic membranes collected in the final days of incubation and 1 week post-hatch. RNA was extracted using Trizol and qRT-PCR was utilized to compare differences during embryo development within and across species., Results: The expression of the IGF mRNA varied between species in the final days of incubation in the amniotic and allantoic membranes. The turkey had higher (0.38-1.72 log) transcript abundance of IGF-I and IGF-II in the amnion and allantois compared to the chicken and duck. Evaluating the mRNA abundance within the chicken duodenum, jejunum, and ileum, the duodenum had the lowest expression of IGF-I and IGF-II (P<0.05) at day -4 of incubation compared to the jejunum and ileum. Focusing on differences in jejunal IGF expression among the three species, the turkey had the lowest IGF-I abundance at day -4 of incubation and highest IGF-I abundance at day of hatch (P<0.05). Transcript abundance of both IGF-II and IGF-R was highest in the turkey at day of hatch and day 1 post-hatch compared to the duck and chicken. The whole tissue versus the mucosal expression of the IGF mRNA abundance was evaluated during the post-hatch period. Duodenal, jejunal, and ileal segments had higher IGF-I transcript abundance (P<0.05) at day 1, day 3, and day of hatch, respectively. No differences were observed between segment and mucosa for IGF-II in the post-hatch period. The duodenal and jejunal mucosa IGF-R transcript abundance was greater (P<0.05) at day of hatch compared to the intestinal segment. The duck IGF mRNA in the jejunal mucosa was higher than the whole segment and decreased from day of hatch to day 3 post-hatch while the IGF mRNA abundance increased in the whole segment during the same time period. The turkey IGF-I transcript abundance decreased in both the segment and mucosa following hatch while the IGF-II mRNA expression increased by 1.5 logs from hatch to day 1 post-hatch., Conclusion: The transcript abundance of the IGF axis in the extra-embryonic membranes and gastrointestinal tissue of the developing chicken, duck, and turkey are influenced by embryonic age and species. A better understanding of the IGF axis in the small intestine during embryonic development may allow for increasing the optimal growth of both the gastrointestinal tract and the neonate.

The first wave of erythropoiesis in amniotic animals generates all primitive erythrocytes and takes place exclusively in yolk sac mesoderm. It is less clear, however, to what extent and for how long the yolk sac contributes to the second wave of erythropoiesis which gives rise to definitive erythrocytes for later embryonic and adult use. Here, we examine the initiation, duration, and site of definitive erythrocyte formation in chicken yolk sac. We show that the earliest definitive erythrocytes are generated in yolk sac venous vessels surrounding major arteries at embryonic day (E) 4-4.5, and that mature definitive erythrocytes enter circulating at E4.5-E5. This takes place at a time when yolk sac vasculature remodels extensively to generate paired arterial/venous vessels. The yolk sac remains the predominant site for definitive erythropoiesis from E5 to E10, and continues to generate definitive erythrocytes at least until E15. Similar to primitive erythropoiesis, definitive erythropoiesis in the yolk sac is accompanied by the expression of transcriptional regulators gata1, scl, and lmo2. Furthermore, our data suggest that one main source of definitive erythropoietic cells is the pre-existing vascular endothelial cells. It remains unclear whether yolk sac derived hematopoietic progenitors that do not undergo erythropoiesis in the yolk sac may take up intraembryonic niches and contribute to erythropoietic stem cell population after hatching., (Copyright (c) 2008 Wiley-Liss, Inc.)

The effect of acute hypoxia (10% O2 for 30 min) on the rate of amnion rhythmic contractions and heart rate (HR) was studied in two age groups of European pond turtle (Emys orbicularis) embryos, on days 19-27 and 37-43 of incubation (30-40 and 60-70% of the period until hatching). Under the control conditions, the two age groups of embryos did not differ from each other in either parameter. Hypoxia did not affect significantly the amnion contraction rate but decreased the HR. The time course of the HR during hypoxia depended on the embryo age. The mean HR in the first group of embryos was 8% decreased by minutes 10-14 of hypoxia and did not change afterwards; in the second group, it was 18% decreased by minutes 3-8 and then partly or completely restored before the end of hypoxic exposure. It has been assumed that the capacity of European pond turtle embryos for restoring the normal HR when exposed to acute hypoxia during the second half of embryogenesis is related to the development of neurohumoral control mechanisms.

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M1, methoctramine for M2, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M3, and tropicamide for M4 receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC50), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 microM), activator of adenylate cyclase (AC), was unidirectional with beta-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC- or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M3 acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M3 acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M3-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.

Extraembryonic development is familiar to mouse researchers, but the term is largely unknown among insect developmental geneticists. This is not surprising, as the model system Drosophila melanogaster has an extremely reduced extraembryonic component, the amnioserosa. In contrast, most insects retain the ancestral complement of two distinct extraembryonic membranes, amnion and serosa. These membranes are involved in several key morphogenetic events at specific developmental stages. The events of anatrepsis and katatrepsis--collectively referred to as blastokinesis--are specific to hemimetabolous insects. Corresponding events in holometabolous insects are simplified and lack formal names. All insects retain dorsal closure, which has been well studied in Drosophila. This review aims to resurrect both the terminology and awareness of insect extraembryonic development--which were last common currency in the late nineteenth and early twentieth centuries--as a number of recent studies have identified essential components of these events, through RNA interference of developmental genes and ectopic hormonal treatments. As much remains unknown, this topic offers opportunities for research on tissue specification, the regulation of cell shape changes and tissue interactions during morphogenesis, tracing the origins and final fates of cell and tissue lineages, and ascertaining the membranes' functions between morphogenetic events.

Higher cyclorrhaphan flies including Drosophila develop a single extraembryonic epithelium (amnioserosa), which closes the germband dorsally. In most other insects two extraembryonic epithelia, serosa and amnion, line the inner eggshell and the ventral germband, respectively. How the two extraembryonic epithelia evolved into one is unclear. Recent studies have shown that, in the flour beetle Tribolium and in the milkweed bug Oncopeltus, the homeobox gene zerknüllt (zen) controls the fusion of the amnion with the serosa before dorsal closure. To understand the origin of the amnioserosa in evolution, we examined the expression and function of zen in the extraembryonic tissue of lower Cyclorrhapha. We show that Megaselia abdita (Phoridae) and Episyrphus balteatus (Syrphidae) develop a serosa and a dorsal amnion, suggesting that a dorsal amnion preceded the origin of the amnioserosa in evolution. Using Krüppel (Kr) and pannier (pnr) homologues of Megaselia as markers for serosal and amniotic tissue, respectively, we show that after zen RNAi all extraembryonic tissue becomes indistinguishable from amniotic cells, like in Tribolium but unlike in Drosophila, in which zen controls all aspects of extraembryonic development. Compared with Megaselia and Episyrphus, zen expression in Drosophila is extended to cells that form the amnion in lower Cyclorrhapha and is down-regulated at the developmental stage, when serosa cells in lower Cyclorrhapha begin to expand. These expression differences between species with distinct extraembryonic tissue organizations and the conserved requirement of zen for serosa development suggest that the origin of an amnioserosa-like epithelium was accompanied by expression changes of zen.

The rate of oxygen consumption (V(O2)), embryo mass, distribution and mass of the chorioallantoic membrane (CAM), heart rate (HR), heart mass, and amnion rhythmic contractions (ARC) were studied in eggs of the European pond turtle (Emys orbicularis) incubated at 28 degrees C for 62.5+/-0.3 days. The V(O2) rapidly increased beginning from incubation day 19 (D19) to a maximum on D50 and then decreased until pipping. The rapid V(O2) rise was correlated with an increase in the CAM surface and mass, heart mass, and ARC amplitude, whereas the functional parameters such as HR and ARC frequency remained unchanged. The drop in V(O2) before pipping was accompanied by a decrease in HR, while the heart and CAM masses were almost constant. In the cases of short-term temperature deviations of +/-3 degrees C from 28 degrees C, changes in (O2) were significant until D50 and nonsignificant after that, the changes in ARC frequency and HR being significant at all stages studied. Thus, the developmental V(O2) changes were contributed mainly by the slow morphogenetic processes during D19-D50, whereas changes in functional parameters began to play a role at later stages. The response to temperature fluctuations was mediated by a rapid change in functional parameters at all these stages.

Somites are segments of paraxial mesoderm that give rise to a multitude of tissues in the vertebrate embryo. Many decades of intensive research have provided a wealth of data on the complex molecular interactions leading to the formation of various somitic derivatives. In this review, we focus on the crucial role of the somites in building the body wall and limbs of amniote embryos. We give an overview on the current knowledge on the specification and differentiation of somitic cell lineages leading to the development of the vertebral column, skeletal muscle, connective tissue, meninges, and vessel endothelium, and highlight the importance of the somites in establishing the metameric pattern of the vertebrate body., ((c) 2007 Wiley-Liss, Inc.)

Somitic and head mesoderm contribute to cartilage and bone and deliver the entire skeletal musculature. Studies on avian somite patterning and cell differentiation led to the view that these processes depend solely on cues from surrounding tissues. However, evidence is accumulating that some developmental decisions depend on information within the somitic tissue itself. Moreover, recent studies established that head and somitic mesoderm, though delivering the same tissue types, are set up to follow their own, distinct developmental programmes. With a particular focus on the chicken embryo, we review the current understanding of how extrinsic signalling, operating in a framework of intrinsically regulated constraints, controls paraxial mesoderm patterning and cell differentiation., ((c) 2007 Wiley-Liss, Inc.)