Time space and single-cell resolved tissue lineage trajectories and laterality of body plan at gastrulation.

Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic molecular trajectories of cell lineages across a series of developmental stages of gastrulation. Through computational modeling, we constructed at single-cell resolution, a spatio-temporal transcriptome of cell populations in the germ-layers of gastrula-stage mouse embryos. This molecular atlas enables the inference of molecular network activity underpinning the specification and differentiation of the germ-layer tissue lineages. Heterogeneity analysis of cellular composition at defined positions in the epiblast revealed progressive diversification of cell types. The single-cell transcriptome revealed an enhanced BMP signaling activity in the right-side mesoderm of late-gastrulation embryo. Perturbation of asymmetric BMP signaling activity at late gastrulation led to randomization of left-right molecular asymmetry in the lateral mesoderm of early-somite-stage embryo. These findings indicate the asymmetric BMP activity during gastrulation may be critical for the symmetry breaking process.

SWI/SNF complexes are required for retinal pigmented epithelium differentiation and for the inhibition of cell proliferation and neural differentiation programs.

During embryonic development, tissue-specific transcription factors and chromatin remodelers function together to ensure gradual, coordinated differentiation of multiple lineages. Here, we define this regulatory interplay in the developing retinal pigmented epithelium (RPE), a neuroectodermal lineage essential for the development, function and maintenance of the adjacent retina. We present a high-resolution spatial transcriptomic atlas of the developing mouse RPE and the adjacent ocular mesenchyme obtained by geographical position sequencing (Geo-seq) of a single developmental stage of the eye that encompasses young and more mature ocular progenitors. These transcriptomic data, available online, reveal the key transcription factors and their gene regulatory networks during RPE and ocular mesenchyme differentiation. Moreover, conditional inactivation followed by Geo-seq revealed that this differentiation program is dependent on the activity of SWI/SNF complexes, shown here to control the expression and activity of RPE transcription factors and, at the same time, inhibit neural progenitor and cell proliferation genes. The findings reveal the roles of the SWI/SNF complexes in controlling the intersection between RPE and neural cell fates and the coupling of cell-cycle exit and differentiation.

Three-dimensional molecular architecture of mouse organogenesis.

Mammalian embryos exhibit sophisticated cellular patterning that is intricately orchestrated at both molecular and cellular level. It has recently become apparent that cells within the animal body display significant heterogeneity, both in terms of their cellular properties and spatial distributions. However, current spatial transcriptomic profiling either lacks three-dimensional representation or is limited in its ability to capture the complexity of embryonic tissues and organs. Here, we present a spatial transcriptomic atlas of all major organs at embryonic day 13.5 in the mouse embryo, and provide a three-dimensional rendering of molecular regulation for embryonic patterning with stacked sections. By integrating the spatial atlas with corresponding single-cell transcriptomic data, we offer a detailed molecular annotation of the dynamic nature of organ development, spatial cellular interactions, embryonic axes, and divergence of cell fates that underlie mammalian development, which would pave the way for precise organ engineering and stem cell-based regenerative medicine.

Base editing-mediated one-step inactivation of the Dnmt gene family reveals critical roles of DNA methylation during mouse gastrulation.

During embryo development, DNA methylation is established by DNMT3A/3B and subsequently maintained by DNMT1. While much research has been done in this field, the functional significance of DNA methylation in embryogenesis remains unknown. Here, we establish a system of simultaneous inactivation of multiple endogenous genes in zygotes through screening for base editors that can efficiently introduce a stop codon. Embryos with mutations in Dnmts and/or Tets can be generated in one step with IMGZ. Dnmt-null embryos display gastrulation failure at E7.5. Interestingly, although DNA methylation is absent, gastrulation-related pathways are down-regulated in Dnmt-null embryos. Moreover, DNMT1, DNMT3A, and DNMT3B are critical for gastrulation, and their functions are independent of TET proteins. Hypermethylation can be sustained by either DNMT1 or DNMT3A/3B at some promoters, which are related to the suppression of miRNAs. The introduction of a single mutant allele of six miRNAs and paternal IG-DMR partially restores primitive streak elongation in Dnmt-null embryos. Thus, our results unveil an epigenetic correlation between promoter methylation and suppression of miRNA expression for gastrulation and demonstrate that IMGZ can accelerate deciphering the functions of multiple genes in vivo.

Simultaneous profiling of spatial gene expression and chromatin accessibility during mouse brain development.

The brain is a complex tissue whose function relies on coordinated anatomical and molecular features. However, the molecular annotation of the spatial organization of the brain is currently insufficient. Here, we describe microfluidic indexing-based spatial assay for transposase-accessible chromatin and RNA-sequencing (MISAR-seq), a method for spatially resolved joint profiling of chromatin accessibility and gene expression. By applying MISAR-seq to the developing mouse brain, we study tissue organization and spatiotemporal regulatory logics during mouse brain development.

Lung development and regeneration: newly defined cell types and progenitor status.

The lung is the most critical organ of the respiratory system supporting gas exchange. Constant interaction with the external environment makes the lung vulnerable to injury. Thus, a deeper understanding of cellular and molecular processes underlying lung development programs and evaluation of progenitor status within the lung is an essential part of lung regenerative medicine. In this review, we aim to discuss the current understanding of lung development process and regenerative capability. We highlight the advances brought by multi-omics approaches, single-cell transcriptome, in particular, that can help us further dissect the cellular player and molecular signaling underlying those processes.

Spatial molecular anatomy of germ layers in the gastrulating cynomolgus monkey embryo.

During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors, and germ layer morphogenesis. While the mouse model has been instrumental for understanding mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a spatial and temporal survey of the molecular dynamics of cell types populating the non-human primate embryos during gastrulation. We reconstructed three-dimensional digital models from serial sections of cynomolgus monkey (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21. Spatial transcriptomics identifies gene expression profiles unique to the germ layers. Cross-species comparison reveals a developmental coordinate of germ layer segregation between mouse and primates, and species-specific transcription programs during gastrulation. These findings offer insights into evolutionarily conserved and divergent processes during mammalian gastrulation.

Embryonic vascular establishment requires protein C receptor-expressing endothelial progenitors.

Vascular establishment is one of the early events in embryogenesis. It is believed that vessel-initiating endothelial progenitors cluster to form the first primitive vessel. Understanding the molecular identity of these progenitors is crucial in order to elucidate lineage hierarchy. In this study, we identify protein C receptor (Procr) as an endothelial progenitor marker and investigate the role of Procr+ progenitors during embryonic vascular development. Using a ProcrmGFP-2A-lacZ reporter, we reveal a much earlier Procr expression (embryonic day 7.5) than previously acknowledged (embryonic day 13.5). Genetic fate-mapping experiments using ProcrCre and ProcrCreER demonstrate that Procr+ cells give rise to blood vessels throughout the entire embryo proper. Single-cell RNA-sequencing analyses place Procr+ cells at the start of endothelial commitment and maturation. Furthermore, targeted ablation of Procr+ cells results in failure of vessel formation and early embryonic lethality. Notably, genetic fate mapping and scRNA-seq pseudotime analysis support the view that Procr+ progenitors can give rise to hemogenic endothelium. In this study, we establish a Procr expression timeline and identify Procr+ vessel-initiating progenitors, and demonstrate their indispensable role in establishment of the vasculature during embryo development.

RNA helicase DDX5 acts as a critical regulator for survival of neonatal mouse gonocytes.

OBJECTIVES: Mammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD-box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes. MATERIALS AND METHODS: Germ cell-specific Ddx5 knockout (Ddx5-/- ) mice were generated. The morphology of testes and epididymides and fertility in both wild-type and Ddx5-/- mice were analysed. Single-cell RNA sequencing (scRNA-seq) was used to profile the transcriptome in testes from wild-type and Ddx5-/- mice at postnatal day (P) 2. Dysregulated genes were validated by single-cell qRT-PCR and immunofluorescent staining. RESULTS: In male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell-specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5-/- mice from E18.5 to P6. Single-cell transcriptome analysis showed that genes involved in cell cycle and glial cell line-derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5-deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes. CONCLUSIONS: Our study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility.

M-CSF, IL-6, and TGF-ß promote generation of a new subset of tissue repair macrophage for traumatic brain injury recovery.

Traumatic brain injury (TBI) leads to high mortality rate. We aimed to identify the key cytokines favoring TBI repair and found that patients with TBI with a better outcome robustly increased concentrations of macrophage colony-stimulating factor, interleukin-6, and transforming growth factor-ß (termed M6T) in cerebrospinal fluid or plasma. Using TBI mice, we identified that M2-like macrophage, microglia, and endothelial cell were major sources to produce M6T. Together with the in vivo tracking of mCherry+ macrophages in zebrafish models, we confirmed that M6T treatment accelerated blood-borne macrophage infiltration and polarization toward a subset of tissue repair macrophages that expressed similar genes as microglia for neuroprotection, angiogenesis and cell migration. M6T therapy in TBI mice and zebrafish improved neurological function while blocking M6T-exacerbated brain injury. Considering low concentrations of M6T in some patients with poor prognostic, M6T treatment might repair TBI via generating a previously unidentified subset of tissue repair macrophages.

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.

Using Single-Cell and Spatial Transcriptomes to Understand Stem Cell Lineage Specification During Early Embryo Development.

Embryonic development and stem cell differentiation provide a paradigm to understand the molecular regulation of coordinated cell fate determination and the architecture of tissue patterning. Emerging technologies such as single-cell RNA sequencing and spatial transcriptomics are opening new avenues to dissect cell organization, the divergence of morphological and molecular properties, and lineage allocation. Rapid advances in experimental and computational tools have enabled researchers to make many discoveries and revisit old hypotheses. In this review, we describe the use of single-cell RNA sequencing in studies of molecular trajectories and gene regulation networks for stem cell lineages, while highlighting the integratedexperimental and computational analysis of single-cell and spatial transcriptomes in the molecular annotation of tissue lineages and development during postimplantation gastrulation.

C-KIT Expression Distinguishes Fetal from Postnatal Skeletal Progenitors.

Hematopoietic stem cells (HSCs) and skeletal stem cells (SSCs) cohabit in the bone marrow. KITL (C-KIT ligand) from LEPR+ adult bone marrow stromal cells is pivotal for HSC maintenance. In contrast, it remains unclear whether KITL/C-KIT signaling also regulates SSCs. Here, we lineage traced C-KIT+ cells and found that C-KIT was expressed by fetal, but not postnatal skeletal progenitors. Fetal C-KIT+ cells gave rise to 20% of LEPR+ stromal cells in adult bone marrow, forming nearly half of all osteoblasts. Disruption of mTOR signaling in fetal C-KIT+ cells impaired bone formation. Notably, conditional deletion of Kitl from PRX1+ fetal bone marrow stromal cells, but not LEPR+ adult bone marrow stromal cells, significantly increased bone formation. Thus, our work identified C-KIT+ skeletal progenitors as an important source of bones formed during development.

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.

Dissecting primate early post-implantation development using long-term in vitro embryo culture.

The transition from peri-implantation to gastrulation in mammals entails the specification and organization of the lineage progenitors into a body plan. Technical and ethical challenges have limited understanding of the cellular and molecular mechanisms that underlie this transition. We established a culture system that enabled the development of cynomolgus monkey embryos in vitro for up to 20 days. Cultured embryos underwent key primate developmental stages, including lineage segregation, bilaminar disc formation, amniotic and yolk sac cavitation, and primordial germ cell-like cell (PGCLC) differentiation. Single-cell RNA-sequencing analysis revealed development trajectories of primitive endoderm, trophectoderm, epiblast lineages, and PGCLCs. Analysis of single-cell chromatin accessibility identified transcription factors specifying each cell type. Our results reveal critical developmental events and complex molecular mechanisms underlying nonhuman primate embryogenesis in the early postimplantation period, with possible relevance to human development.

Human Neural Stem Cells Reinforce Hippocampal Synaptic Network and Rescue Cognitive Deficits in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by memory impairments in its earliest clinical phase. The synaptic loss and dysfunction leading to failures of synaptic networks in AD brain directly cause cognitive deficits of patient. However, it remains unclear whether the synaptic networks in AD brain could be repaired. In this study, we generated functional human induced neural progenitor/stem cells (iNPCs) that had been transplanted into the hippocampus of immunodeficient wild-type and AD mice. The grafted human iNPCs efficiently differentiated into neurons that displayed long-term survival, progressively acquired mature membrane properties, formed graft-host synaptic connections with mouse neurons and functionally integrated into local synaptic circuits, which eventually reinforced and repaired the neural networks of host hippocampus. Consequently, AD mice with human iNPCs exhibited enhanced synaptic plasticity and improved cognitive abilities. Together, our results suggest that restoring synaptic failures by stem cells might provide new directions for the development of novel treatments for human AD.

A secreted microRNA disrupts autophagy in distinct tissues of Caenorhabditis elegans upon ageing.

Macroautophagy, a key player in protein quality control, is proposed to be systematically impaired in distinct tissues and causes coordinated disruption of protein homeostasis and ageing throughout the body. Although tissue-specific changes in autophagy and ageing have been extensively explored, the mechanism underlying the inter-tissue regulation of autophagy with ageing is poorly understood. Here, we show that a secreted microRNA, mir-83/miR-29, controls the age-related decrease in macroautophagy across tissues in Caenorhabditis elegans. Upregulated in the intestine by hsf-1/HSF1 with age, mir-83 is transported across tissues potentially via extracellular vesicles and disrupts macroautophagy by suppressing CUP-5/MCOLN, a vital autophagy regulator, autonomously in the intestine as well as non-autonomously in body wall muscle. Mutating mir-83 thereby enhances macroautophagy in different tissues, promoting protein homeostasis and longevity. These findings thus identify a microRNA-based mechanism to coordinate the decreasing macroautophagy in various tissues with age.

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.