Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids.

The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.

Memory CD4+ T cells are generated in the human fetal intestine.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.

Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. 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.

Fetal germ cell development in humans, a link with infertility.

Gametes are cells that have the unique ability to give rise to new individuals as well as transmit (epi)genetic information across generations. Generation of functionally competent gametes, oocytes and sperm cells, depends to some extent on several fundamental processes that occur during fetal development. Direct studies on human fetal germ cells remain hindered by ethical considerations and inaccessibility to human fetal material. Therefore, the majority of our current knowledge of germ cell development still comes from an invaluable body of research performed using different mammalian species. During the last decade, our understanding of human fetal germ cells has increased due to the successful use of human pluripotent stem cells to model aspects of human early gametogenesis and advancements on single-cell omics. Together, this has contributed to determine the cell types and associated molecular signatures in the developing human gonads. In this review, we will put in perspective the knowledge obtained from several mammalian models (mouse, monkey, pig). Moreover, we will discuss the main events during human fetal (female) early gametogenesis and how the dysregulation of this highly complex and lengthy process can link to infertility later in life.

Transcriptional progression during meiotic prophase I reveals sex-specific features and X chromosome dynamics in human fetal female germline.

During gametogenesis in mammals, meiosis ensures the production of haploid gametes. The timing and length of meiosis to produce female and male gametes differ considerably. In contrast to males, meiotic prophase I in females initiates during development. Hence, the knowledge regarding progression through meiotic prophase I is mainly focused on human male spermatogenesis and female oocyte maturation during adulthood. Therefore, it remains unclear how the different stages of meiotic prophase I between human oogenesis and spermatogenesis compare. Analysis of single-cell transcriptomics data from human fetal germ cells (FGC) allowed us to identify the molecular signatures of female meiotic prophase I stages leptotene, zygotene, pachytene and diplotene. We have compared those between male and female germ cells in similar stages of meiotic prophase I and revealed conserved and specific features between sexes. We identified not only key players involved in the process of meiosis, but also highlighted the molecular components that could be responsible for changes in cellular morphology that occur during this developmental period, when the female FGC acquire their typical (sex-specific) oocyte shape as well as sex-differences in the regulation of DNA methylation. Analysis of X-linked expression between sexes during meiotic prophase I suggested a transient X-linked enrichment during female pachytene, that contrasts with the meiotic sex chromosome inactivation in males. Our study of the events that take place during meiotic prophase I provide a better understanding not only of female meiosis during development, but also highlights biomarkers that can be used to study infertility and offers insights in germline sex dimorphism in humans.

Classification of Atretic Small Antral Follicles in the Human Ovary.

The reproductive lifespan in humans is regulated by a delicate cyclical balance between follicular recruitment and atresia in the ovary. The majority of the small antral follicles present in the ovary are progressively lost through atresia without reaching dominance, but this process remains largely underexplored. In our study, we investigated the characteristics of atretic small antral follicles and proposed a classification system based on molecular changes observed in granulosa cells, theca cells, and extracellular matrix deposition. Our findings revealed that atresia spreads in the follicle with wave-like dynamics, initiating away from the cumulus granulosa cells. We also observed an enrichment of CD68+ macrophages in the antrum during the progression of follicular atresia. This work not only provides criteria for classifying three stages of follicular atresia in small antral follicles in the human ovary but also serves as a foundation for understanding follicular degeneration and ultimately preventing or treating premature ovarian failure. Understanding follicular remodeling in the ovary could provide a means to increase the number of usable follicles and delay the depletion of the follicular reserve, increasing the reproductive lifespan.

Rethinking organoid technology through bioengineering.

In recent years considerable progress has been made in the development of faithful procedures for the differentiation of human pluripotent stem cells (hPSCs). An important step in this direction has also been the derivation of organoids. This technology generally relies on traditional three-dimensional culture techniques that exploit cell-autonomous self-organization responses of hPSCs with minimal control over the external inputs supplied to the system. The convergence of stem cell biology and bioengineering offers the possibility to provide these stimuli in a controlled fashion, resulting in the development of naturally inspired approaches to overcome major limitations of this nascent technology. Based on the current developments, we emphasize the achievements and ongoing challenges of bringing together hPSC organoid differentiation, bioengineering and ethics. This Review underlines the need for providing engineering solutions to gain control of self-organization and functionality of hPSC-derived organoids. We expect that this knowledge will guide the community to generate higher-grade hPSC-derived organoids for further applications in developmental biology, drug screening, disease modelling and personalized medicine.

Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells.

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.

Single-cell transcriptomics reveals gene expression dynamics of human fetal kidney development.

The current understanding of mammalian kidney development is largely based on mouse models. Recent landmark studies revealed pervasive differences in renal embryogenesis between mouse and human. The scarcity of detailed gene expression data in humans therefore hampers a thorough understanding of human kidney development and the possible developmental origin of kidney diseases. In this paper, we present a single-cell transcriptomics study of the human fetal kidney. We identified 22 cell types and a host of marker genes. Comparison of samples from different developmental ages revealed continuous gene expression changes in podocytes. To demonstrate the usefulness of our data set, we explored the heterogeneity of the nephrogenic niche, localized podocyte precursors, and confirmed disease-associated marker genes. With close to 18,000 renal cells from five different developmental ages, this study provides a rich resource for the elucidation of human kidney development, easily accessible through an interactive web application.

Amniotic ectoderm expansion in mouse occurs via distinct modes and requires SMAD5-mediated signalling.

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.

Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.

The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.

Single-Cell Transcriptomics Analysis of Human Small Antral Follicles.

Human ovarian folliculogenesis is a highly regulated and complex process. Characterization of follicular cell signatures during this dynamic process is important to understand follicle fate (to grow, become dominant, or undergo atresia). The transcriptional signature of human oocytes and granulosa cells (GCs) in early-growing and ovulatory follicles have been previously described; however, that of oocytes with surrounding GCs in small antral follicles have not been studied yet. Here, we have generated a unique dataset of single-cell transcriptomics (SmartSeq2) consisting of the oocyte with surrounding GCs from several individual (non-dominant) small antral follicles isolated from adult human ovaries. We have identified two main types of (healthy) follicles, with a distinct oocyte and GC signature. Using the CellphoneDB algorithm, we then investigated the bi-directional ligand-receptor interactions regarding the transforming growth factor-ß (TGFß)/bone morphogenetic protein (BMP), wingless-type (MMTV)-integration site (WNT), NOTCH, and receptor tyrosine kinases (RTK) signaling pathways between oocyte and GCs within each antral follicle type. Our work not only revealed the diversity of small antral follicles, but also contributes to fill the gap in mapping the molecular landscape of human folliculogenesis and oogenesis.

Human blastocyst outgrowths recapitulate primordial germ cell specification events.

Our current knowledge of the mechanisms leading to human primordial germ cell (PGC) specification stems solely from differentiation experiments starting from human pluripotent stem cells. However, information regarding the origin of PGCs in vivo remains obscure. Here we apply an improved system for extended in vitro culture of human embryos to investigate the presence of PGC-like cells (PGCLCs) 12 days post fertilization (dpf). Good quality blastocysts (n = 141) were plated at 6 dpf and maintained in hypoxia, in medium supplemented with Activin A until 12 dpf. We primarily reveal that 12 dpf outgrowths recapitulate human peri-implantation events and demonstrate that blastocyst quality significantly impacts both embryo viability at 12 dpf, as well as the presence of POU5F1+ cells within viable outgrowths. Moreover, detailed examination of 12 dpf blastocyst outgrowths revealed a population of POU5F1+, SOX2- and SOX17+ cells that may correspond to PGCLCs, alongside POU5F1+ epiblast-like cells and GATA6+ endoderm-like cells. Our findings suggest that, in human, PGC precursors may become specified within the epiblast and migrate either transiently to the extra-embryonic mesoderm or directly to the dorsal part of the yolk sac endoderm around 12 dpf. This is a descriptive analysis and as such the conclusion that POU5F1+ and SOX17+ cells represent bona fide PGCs can only be considered as preliminary. In the future, other PGC markers may be used to further validate the observed cell populations. Overall, our findings provide insights into the origin of the human germline and may serve as a foundation to further unravel the molecular mechanisms governing PGC specification in human.

Transcriptome of human foetal heart compared with cardiomyocytes from pluripotent stem cells.

Differentiated derivatives of human pluripotent stem cells (hPSCs) are often considered immature because they resemble foetal cells more than adult, with hPSC-derived cardiomyocytes (hPSC-CMs) being no exception. Many functional features of these cardiomyocytes, such as their cell morphology, electrophysiological characteristics, sarcomere organization and contraction force, are underdeveloped compared with adult cardiomyocytes. However, relatively little is known about how their gene expression profiles compare with the human foetal heart, in part because of the paucity of data on the human foetal heart at different stages of development. Here, we collected samples of matched ventricles and atria from human foetuses during the first and second trimester of development. This presented a rare opportunity to perform gene expression analysis on the individual chambers of the heart at various stages of development, allowing us to identify not only genes involved in the formation of the heart, but also specific genes upregulated in each of the four chambers and at different stages of development. The data showed that hPSC-CMs had a gene expression profile similar to first trimester foetal heart, but after culture in conditions shown previously to induce maturation, they cluster closer to the second trimester foetal heart samples. In summary, we demonstrate how the gene expression profiles of human foetal heart samples can be used for benchmarking hPSC-CMs and also contribute to determining their equivalent stage of development.

Characterization of migratory primordial germ cells in the aorta-gonad-mesonephros of a 4.5-week-old human embryo: a toolbox to evaluate in vitro early gametogenesis.

STUDY QUESTION: Which set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)? STUDY FINDING: We validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12-13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM). WHAT IS KNOWN ALREADY: The dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well. STUDY DESIGN, SIZE, DURATION: This immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development. PARTICIPANTS/MATERIALS, SETTING, METHODS: We have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12-13). The human material used was anonymously donated with informed consent from elective abortions without medical indication. MAIN RESULTS AND THE ROLE OF CHANCE: We observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs. LARGE SCALE DATA: Non-applicable. LIMITATIONS REASONS FOR CAUTION: Material to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available. WIDER IMPLICATIONS OF THE FINDINGS: Most of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the expression of 31 different markers in paraffin sections of a CS12-13 embryo. Our results will help to set up a toolbox of markers to evaluate protocols to induce hPGCLCs in vitro. STUDY FUNDING AND COMPETING INTEREST(S): M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011] and S.M.C.S.L. was funded by the Interuniversity Attraction Poles (IAP, P7/07) and the European Research Council Consolidator (ERC-CoG-725722-OVOGROWTH). The authors declare no conflict of interest.

Human-specific subcellular compartmentalization of P-element induced wimpy testis-like (PIWIL) granules during germ cell development and spermatogenesis.

STUDY QUESTION: What is the dynamics of expression of P-element induced wimpy testis-like (PIWIL) proteins in the germline during human fetal development and spermatogenesis? SUMMARY ANSWER: PIWIL1, PIWIL2, PIWIL3 and PIWIL4 were expressed in a sex-specific fashion in human germ cells (GC) during development and adulthood. PIWILs showed a mutually exclusive pattern of subcellular localization. PIWILs were present in the intermitochondrial cement and a single large granule in meiotic GC and their expression was different from that observed in mice, highlighting species-differences. WHAT IS KNOWN ALREADY: In mice, PIWIL proteins play prominent roles in male infertility. PIWIL mouse mutants show either post-meiotic arrest at the round spermatid stage (PIWIL1) or arrest at the zygotene-pachytene stage of meiosis I (PIWIL2 and PIWIL4) in males, while females remain fertile. Recent studies have reported a robust piRNA pool in human fetal ovary. STUDY DESIGN, SIZE, DURATION: This is a qualitative analysis of PIWILs expression in paraffin-embedded fetal human male (N = 8), female gonads (N = 6) and adult testes (N = 5), and bioinformatics analysis of online available single-cell transcriptomics data of human fetal germ cells (n = 242). PARTICIPANTS/MATERIALS, SETTING, METHODS: Human fetal gonads from elective abortion without medical indication and adult testes biopsies were donated for research with informed consent. Samples were fixed, paraffin-embedded and analyzed by immunofluorescence to study the temporal and cellular localization of PIWIL1, PIWIL2, PIWIL3 and PIWIL4. MAIN RESULTS AND THE ROLE OF CHANCE: PIWIL1, PIWIL2 and PIWIL4 showed a mutually exclusive pattern of subcellular localization, particularly in female oocytes. To our surprise, PIWIL1 immunostaining revealed the presence of a single dense paranuclear body, resembling the chromatoid body of haploid spermatocytes, in meiotic oocytes. Moreover, in contrast to mice, PIWIL4, but not PIWIL2, localized to the intermitochondrial cement. PIWIL3 was not expressed in GC during development. The upregulation of PIWIL transcripts correlated with the transcription of markers associated with piRNAs biogenesis like the TDRDs and HENMT1 in fetal GC. LARGE SCALE DATA: Non-applicable. LIMITATIONS, REASONS FOR CAUTION: This study is limited by the restricted number of samples and consequently stages analyzed. WIDER IMPLICATIONS OF THE FINDINGS: In the germline, PIWILs ensure the integrity of the human genome protecting it from 'parasitic sequences'. This study offers novel insights on the expression dynamics of PIWILs during the window of epigenetic remodeling and meiosis, and highlights important differences between humans and mice, which may prove particularly important to understand causes of infertility and improve both diagnosis and treatment in humans. STUDY FUNDING/COMPETING INTEREST(S): M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011]; N.H. by China Scholarship Council (CSC) [No. 201307040026] and F.W. by Medical Personnel Training Abroad Project of Henan Province [No. 2015022] and S.M.C.d.S.L. by the Netherlands Organization of Scientific Research (NWO) [ASPASIA 015.007.037] and the Interuniversity Attraction Poles-Phase VII [IUAP/PAI P7/14]. The authors have no conflicts of interest to declare.

Germline development in amniotes: A paradigm shift in primordial germ cell specification.

In the field of germline development in amniote vertebrates, primordial germ cell (PGC) specification in birds and reptiles remains controversial. Avians are believed to adopt a predetermination or maternal specification mode of PGC formation, contrary to an inductive mode employed by mammals and, supposedly, reptiles. Here, we revisit and review some key aspects of PGC development that channelled the current subdivision, and challenge the position of birds and reptiles as well as the 'binary' evolutionary model of PGC development in vertebrates. We propose an alternative view on PGC specification where germ plasm plays a role in laying the foundation for the formation of PGC precursors (pPGC), but not necessarily of PGCs. Moreover, inductive mechanisms may be necessary for the transition from pPGCs to PGCs. Within this framework, the implementation of data from birds and reptiles could provide new insights on the evolution of PGC specification in amniotes.

DNA Methylation Landscapes of Human Fetal Development.

Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.

A conserved role for non-neural ectoderm cells in early neural development.

During the early steps of head development, ectodermal patterning leads to the emergence of distinct non-neural and neural progenitor cells. The induction of the preplacodal ectoderm and the neural crest depends on well-studied signalling interactions between the non-neural ectoderm fated to become epidermis and the prospective neural plate. By contrast, the involvement of the non-neural ectoderm in the morphogenetic events leading to the development and patterning of the central nervous system has been studied less extensively. Here, we show that the removal of the rostral non-neural ectoderm abutting the prospective neural plate at late gastrulation stage leads, in mouse and chick embryos, to morphological defects in forebrain and craniofacial tissues. In particular, this ablation compromises the development of the telencephalon without affecting that of the diencephalon. Further investigations of ablated mouse embryos established that signalling centres crucial for forebrain regionalization, namely the axial mesendoderm and the anterior neural ridge, form normally. Moreover, changes in cell death or cell proliferation could not explain the specific loss of telencephalic tissue. Finally, we provide evidence that the removal of rostral tissues triggers misregulation of the BMP, WNT and FGF signalling pathways that may affect telencephalon development. This study opens new perspectives on the role of the neural/non-neural interface and reveals its functional relevance across higher vertebrates.