Mouse organogenesis atlas at single-cell resolution.

The generation of spatial transcriptomes of whole embryo has been limited in scale and resolution due to various technological restrictions. In this issue of Cell, Chen et al. introduce a DNA nanoball-based sample-capture technology for spatial transcriptome analysis to generate a molecular atlas of mouse organogenesis at single-cell resolution.

Human developmental biology - a global perspective.

In the companion Perspective 'Past and future of human developmental biology' (Hopwood, 2024), historian Nick Hopwood proposes that the field of human developmental biology has gone through periods of attention and neglect. Development invited researchers from the field to respond to this idea. In this article, published to coincide with the 10th anniversary of Development's 'From Stem Cells to Human Development' meeting, researchers from eight countries comment on how they believe their local legal, political, regulatory, societal and technological frameworks are influencing the field's trajectory.

Technical challenges of studying early human development.

Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.

Author Correction: Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

During post-implantation development of the mouse embryo, descendants of the inner cell mass in the early epiblast transit from the naive to primed pluripotent state1. Concurrently, germ layers are formed and cell lineages are specified, leading to the establishment of the blueprint for embryogenesis. Fate-mapping and lineage-analysis studies have revealed that cells in different regions of the germ layers acquire location-specific cell fates during gastrulation2-5. The regionalization of cell fates preceding the formation of the basic body plan-the mechanisms of which are instrumental for understanding embryonic programming and stem-cell-based translational study-is conserved in vertebrate embryos6-8. However, a genome-wide molecular annotation of lineage segregation and tissue architecture of the post-implantation embryo has yet to be undertaken. Here we report a spatially resolved transcriptome of cell populations at defined positions in the germ layers during development from pre- to late-gastrulation stages. This spatiotemporal transcriptome provides high-resolution digitized in situ gene-expression profiles, reveals the molecular genealogy of tissue lineages and defines the continuum of pluripotency states in time and space. The transcriptome further identifies the networks of molecular determinants that drive lineage specification and tissue patterning, supports a role of Hippo-Yap signalling in germ-layer development and reveals the contribution of visceral endoderm to the endoderm in the early mouse embryo.

Donor-derived Mycoplasma and Ureaplasma infections in lung transplant recipients: A prospective study of donor and recipient respiratory tract screening and recipient outcomes.

Mycoplasma hominis and Ureaplasma species are urogenital mollicutes that can cause serious donor-derived infections in lung transplant recipients. Best practices for mollicute screening remain unknown. We conducted a single-center prospective study analyzing lung transplants performed from October 5, 2020, to September 25, 2021, whereby donor and recipient bronchoalveolar lavage (BAL) samples obtained at time of transplant underwent mollicute screening via culture and polymerase chain reaction (PCR). Of 115 total lung transplants performed, 99 (86%) donors underwent combined mollicute BAL culture and PCR testing. The study cohort included these 99 donors and their matched recipients. In total, 18 (18%) of 99 donors screened positive via culture or PCR. Among recipients, 92 (93%) of 99 had perioperative BAL screening performed, and only 3 (3%) had positive results. After transplant, 9 (9%) recipients developed mollicute infection. Sensitivity of donor screening in predicting recipient mollicute infection was 67% (6/9) via culture and 56% (5/9) via PCR. Positive predictive value for donor culture was 75% (6/8), compared with 33% (5/15) for PCR. Donor screening via culture predicted all serious recipient mollicute infections and had better positive predictive value than PCR; however, neither screening test predicted all mollicute infections. Independent of screening results, clinicians should remain suspicious for posttransplant mollicute infection.

Risk factors, management, and clinical outcomes of invasive Mycoplasma and Ureaplasma infections after lung transplantation.

Mollicute infections, caused by Mycoplasma and Ureaplasma species, are serious complications after lung transplantation; however, understanding of the epidemiology and outcomes of these infections remains limited. We conducted a single-center retrospective study of 1156 consecutive lung transplants performed from 2010-2019. We used log-binomial regression to identify risk factors for infection and analyzed clinical management and outcomes. In total, 27 (2.3%) recipients developed mollicute infection. Donor characteristics independently associated with recipient infection were age ≤40 years (prevalence rate ratio [PRR] 2.6, 95% CI 1.0-6.9), White race (PRR 3.1, 95% CI 1.1-8.8), and purulent secretions on donor bronchoscopy (PRR 2.3, 95% CI 1.1-5.0). Median time to diagnosis was 16 days posttransplant (IQR: 11-26 days). Mollicute-infected recipients were significantly more likely to require prolonged ventilatory support (66.7% vs 21.4%), undergo dialysis (44.4% vs 6.3%), and remain hospitalized ≥30 days (70.4% vs 27.4%) after transplant. One-year posttransplant mortality in mollicute-infected recipients was 12/27 (44%), compared to 148/1129 (13%) in those without infection (P <.0001). Hyperammonemia syndrome occurred in 5/27 (19%) mollicute-infected recipients, of whom 3 (60%) died within 10 weeks posttransplant. This study highlights the morbidity and mortality associated with mollicute infection after lung transplantation and the need for better screening and management protocols.

Specifying mouse embryonic germ cells.

Male germ cells are induced to form from the epiblast of the mouse embryo by a combination of WNT and bone morphogenetic protein signals. Ohinata et al. (2009) now clarify the steps of mouse germ cell formation and use this genetic insight to direct the specification and differentiation of germline progenitor cells in vitro.

The diagnostic accuracy of Gram stain on formalin-fixed paraffin-embedded sections of skin tissue in the diagnosis of bacterial skin infection.

BACKGROUND AND OBJECTIVE: To evaluate the sensitivity, specificity, and likelihood ratios of Gram stain on formalin-fixed, paraffin-embedded (GS-FFPE) sections of skin in diagnosing bacterial skin infection. METHODS: We reviewed a retrospective series of skin specimens reported at our institution wherein histopathological assessment included Gram stain and fresh tissue was concurrently submitted for microscopy and culture. The clinicopathological correlation was the reference standard, whereby the presence of infection was deduced from the final diagnosis in each patient's case notes. RESULTS: Our sample included 168 cases (105 positive for infection). GS-FFPE showed a sensitivity of 0.43 (95% confidence interval 0.29, 0.57), a specificity of 0.98 (0.95, 1.01), a positive likelihood ratio of 21.50 (19.76, 23.24), and a negative likelihood ratio of 0.58 (0.41, 0.75). CONCLUSIONS: GS-FFPE has poor sensitivity, and a negative result should not be used as evidence to exclude infection. In contrast, it has excellent specificity and, unless the pretest probability of infection is very low, a positive result would make infection much more likely. The value of the GS-FFPE lies in cases where sterile tissue was not submitted for microbiological studies, or sterile tissue culture was negative, and there is at least a low-to-moderate pretest probability of infection.

Cellular diversity and lineage trajectory: insights from mouse single cell transcriptomes.

Single cell RNA-sequencing (scRNA-seq) technology has matured to the point that it is possible to generate large single cell atlases of developing mouse embryos. These atlases allow the dissection of developmental cell lineages and molecular changes during embryogenesis. When coupled with single cell technologies for profiling the chromatin landscape, epigenome, proteome and metabolome, and spatial tissue organisation, these scRNA-seq approaches can now collect a large volume of multi-omic data about mouse embryogenesis. In addition, advances in computational techniques have enabled the inference of developmental lineages of differentiating cells, even without explicitly introduced genetic markers. This Spotlight discusses recent advent of single cell experimental and computational methods, and key insights from applying these methods to the study of mouse embryonic development. We highlight challenges in analysing and interpreting these data to complement and expand our knowledge from traditional developmental biology studies in relation to cell identity, diversity and lineage differentiation.

Establishment of mouse expanded potential stem cells.

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

Differential impact of TGFß/SMAD signaling activity elicited by Activin A and Nodal on endoderm differentiation of epiblast stem cells.

Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFß pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGFß family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGFß pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGFß/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGFß/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells.

Dynamics of Wnt activity on the acquisition of ectoderm potency in epiblast stem cells.

During embryogenesis, the stringent regulation of Wnt activity is crucial for the morphogenesis of the head and brain. The loss of function of the Wnt inhibitor Dkk1 results in elevated Wnt activity, loss of ectoderm lineage attributes from the anterior epiblast, and the posteriorisation of anterior germ layer tissue towards the mesendoderm. The modulation of Wnt signalling may therefore be crucial for the allocation of epiblast cells to ectoderm progenitors during gastrulation. To test this hypothesis, we examined the lineage characteristics of epiblast stem cells (EpiSCs) that were derived and maintained under different signalling conditions. We showed that suppression of Wnt activity enhanced the ectoderm propensity of the EpiSCs. Neuroectoderm differentiation of these EpiSCs was further empowered by the robust re-activation of Wnt activity. Therefore, during gastrulation, the tuning of the signalling activities that mediate mesendoderm differentiation is instrumental for the acquisition of ectoderm potency in the epiblast.

Mammalian gastrulation: signalling activity and transcriptional regulation of cell lineage differentiation and germ layer formation.

The interplay of signalling input and downstream transcriptional activity is the key molecular attribute driving the differentiation of germ layer tissue and the specification of cell lineages within each germ layer during gastrulation. This review delves into the current understanding of signalling and transcriptional control of lineage development in the germ layers of mouse embryo and non-human primate embryos during gastrulation and highlights the inter-species conservation and divergence of the cellular and molecular mechanisms of germ layer development in the human embryo.

Transcriptional network dynamics during the progression of pluripotency revealed by integrative statistical learning.

The developmental potential of cells, termed pluripotency, is highly dynamic and progresses through a continuum of naive, formative and primed states. Pluripotency progression of mouse embryonic stem cells (ESCs) from naive to formative and primed state is governed by transcription factors (TFs) and their target genes. Genomic techniques have uncovered a multitude of TF binding sites in ESCs, yet a major challenge lies in identifying target genes from functional binding sites and reconstructing dynamic transcriptional networks underlying pluripotency progression. Here, we integrated time-resolved 'trans-omic' datasets together with TF binding profiles and chromatin conformation data to identify target genes of a panel of TFs. Our analyses revealed that naive TF target genes are more likely to be TFs themselves than those of formative TFs, suggesting denser hierarchies among naive TFs. We also discovered that formative TF target genes are marked by permissive epigenomic signatures in the naive state, indicating that they are poised for expression prior to the initiation of pluripotency transition to the formative state. Finally, our reconstructed transcriptional networks pinpointed the precise timing from naive to formative pluripotency progression and enabled the spatiotemporal mapping of differentiating ESCs to their in vivo counterparts in developing embryos.

Meta-Analysis Identifies BDNF and Novel Common Genes Differently Altered in Cross-Species Models of Rett Syndrome.

Rett syndrome (RTT) is a rare disorder and one of the most abundant causes of intellectual disabilities in females. Single mutations in the gene coding for methyl-CpG-binding protein 2 (MeCP2) are responsible for the disorder. MeCP2 regulates gene expression as a transcriptional regulator as well as through epigenetic imprinting and chromatin condensation. Consequently, numerous biological pathways on multiple levels are influenced. However, the exact molecular pathways from genotype to phenotype are currently not fully elucidated. Treatment of RTT is purely symptomatic as no curative options for RTT have yet to reach the clinic. The paucity of this is mainly due to an incomplete understanding of the underlying pathophysiology of the disorder with no clinically useful common disease drivers, biomarkers, or therapeutic targets being identified. With the premise of identifying universal and robust disease drivers and therapeutic targets, here, we interrogated a range of RTT transcriptomic studies spanning different species, models, and MECP2 mutations. A meta-analysis using RNA sequencing data from brains of RTT mouse models, human post-mortem brain tissue, and patient-derived induced pluripotent stem cell (iPSC) neurons was performed using weighted gene correlation network analysis (WGCNA). This study identified a module of genes common to all datasets with the following ten hub genes driving the expression: ATRX, ADCY7, ADCY9, SOD1, CACNA1A, PLCG1, CCT5, RPS9, BDNF, and MECP2. Here, we discuss the potential benefits of these genes as therapeutic targets.

Detectable Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Human Breast Milk of a Mildly Symptomatic Patient With Coronavirus Disease 2019 (COVID-19).

SARS-CoV-2 is a novel coronavirus and causative pathogen to the pandemic illness COVID-19. Although RNA has been detected in various clinical samples, no reports to date have documented SARS-CoV-2 in human milk. This case report describes an actively breastfeeding patient with COVID-19 infection with detectable viral RNA in human milk.

Ularcirc: visualization and enhanced analysis of circular RNAs via back and canonical forward splicing.

Circular RNAs (circRNA) are a unique class of transcripts that can only be identified from sequence alignments spanning discordant junctions, commonly referred to as backsplice junctions (BSJ). Canonical splicing is also linked with circRNA biogenesis either from the parental transcript or internal to the circRNA, and is not fully utilized in circRNA software. Here we present Ularcirc, a software tool that integrates the visualization of both BSJ and forward splicing junctions and provides downstream analysis of selected circRNA candidates. Ularcirc utilizes the output of CIRI, circExplorer, or raw chimeric output of the STAR aligner and assembles BSJ count table to allow multi-sample analysis. We used Ularcirc to identify and characterize circRNA from public and in-house generated data sets and demonstrate how it can be used to (i) discover novel splicing patterns of parental transcripts, (ii) detect internal splicing patterns of circRNA, and (iii) reveal the complexity of BSJ formation. Furthermore, we identify circRNA that have potential open reading frames longer than their linear sequence. Finally, we detected and validated the presence of a novel class of circRNA generated from ApoA4 transcripts whose BSJ derive from multiple non-canonical splicing sites within coding exons. Ularcirc is accessed via https://github.com/VCCRI/Ularcirc.

Alternative lengthening of telomeres in normal mammalian somatic cells.

Some cancers use alternative lengthening of telomeres (ALT), a mechanism whereby new telomeric DNA is synthesized from a DNA template. To determine whether normal mammalian tissues have ALT activity, we generated a mouse strain containing a DNA tag in a single telomere. We found that the tagged telomere was copied by other telomeres in somatic tissues but not the germline. The tagged telomere was also copied by other telomeres when introgressed into CAST/EiJ mice, which have telomeres more similar in length to those of humans. We conclude that ALT activity occurs in normal mouse somatic tissues.