Postconceptional Weeks Research Articles

High-parametric protein maps reveal the spatial organization in early-developing human lung

The respiratory system, including the lungs, is essential for terrestrial life. While recent research has advanced our understanding of lung development, much still relies on animal models and transcriptome analyses. In this study conducted within the Human Developmental Cell Atlas (HDCA) initiative, we describe the protein-level spatiotemporal organization of the lung during the first trimester of human gestation. Using high-parametric tissue imaging with a 30-plex antibody panel, we analyzed human lung samples from 6 to 13 post-conception weeks, generating data from over 2 million cells across five developmental timepoints. We present a resource detailing spatially resolved cell type composition of the developing human lung, including proliferative states, immune cell patterns, spatial arrangement traits, and their temporal evolution. This represents an extensive single-cell resolved protein-level examination of the developing human lung and provides a valuable resource for further research into the developmental roots of human respiratory health and disease.

A prenatal skin atlas reveals immune regulation of human skin morphogenesis.

Human prenatal skin is populated by innate immune cells, including macrophages, but whether they act solely in immunity or have additional functions in morphogenesis is unclear. Here we assembled a comprehensive multi-omics reference atlas of prenatal human skin (7-17 post-conception weeks), combining single-cell and spatial transcriptomics data, to characterize the microanatomical tissue niches of the skin. This atlas revealed that crosstalk between non-immune and immune cells underpins the formation of hair follicles, is implicated inscarless wound healing and is crucial for skin angiogenesis. We systematically compared a hair-bearing skin organoid (SkO) model derived from human embryonic stem cells and induced pluripotent stem cells to prenatal and adult skin1. The SkO model closely recapitulated in vivo skin epidermal and dermal cell types during hair follicle development and expression of genes implicated in the pathogenesis of genetic hair and skin disorders. However, the SkO model lacked immune cells and had markedly reduced endothelial cell heterogeneity and quantity. Our in vivo prenatal skin cell atlas indicated that macrophages and macrophage-derived growth factors have a role in driving endothelial development. Indeed, vascular network remodelling was enhanced following transfer of autologous macrophages derived from induced pluripotent stem cells into SkO cultures. Innate immune cells are therefore key players in skin morphogenesis beyond their conventional role in immunity, a function they achieve through crosstalk with non-immune cells.

Embryonic macrophages support endocrine commitment during human pancreatic differentiation

Organogenesis is a complex process that relies on a dynamic interplay between extrinsic factors originating from the microenvironment and tissue-specific intrinsic factors. For pancreatic endocrine cells, the local niche consists of acinar and ductal cells as well as neuronal, immune, endothelial, and stromal cells. Hematopoietic cells have been detected in human pancreas as early as 6 post-conception weeks, but whether they play a role during human endocrinogenesis remains unknown. To investigate this, we performed single-nucleus RNA sequencing (snRNA-seq) of the second-trimester human pancreas and identified a wide range of hematopoietic cells, including two distinct subsets of tissue-resident macrophages. Leveraging this discovery, we developed a co-culture system of human embryonic stem cell-derived endocrine-macrophage organoids to model their interaction invitro. Here, we show that macrophages support the differentiation and viability of endocrine cells invitro and enhance tissue engraftment, highlighting their potential role in tissue engineering strategies for diabetes.

Deciphering the impact of PROM1 alternative splicing on human photoreceptor development and maturation

Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity, which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular, photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs), undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy, however its role in photoreceptor maturation and function is unknown. In this study retinal organoids, a valuable model system, were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR, western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%, abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone, inner segment and connecting cilium basal body markers, increased expression of genes associated with stress response and the ubiquitin-proteasome system, and downregulation of autophagy. Importantly, the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context, opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression, corroborating the pathogenesis of late-onset mild maculopathy.

Discovering genes and microRNAs involved in human lung development unveils IGFBP3/miR-34a dynamics and their relevance for alveolar differentiation

BackgroundDuring pseudoglandular stage of the human lung development the primitive bronchial buds are initially conformed by simple tubules lined by endoderm-derived epithelium surrounded by mesenchyme, which will progressively branch into airways and start to form distal epithelial saculles. For first time alveolar type II (AT2) pneumocytes appears. This study aims to characterize the genes and microRNAs involved in this differentiation process and decipher its role in the starting alveolar differentiation.MethodsGene and microRNA profiling was performed in human embryonic lungs from 7 to 12 post conception weeks (pcw). Protein expression location of candidate genes were analyzed by immunofluorescense in embryonic lung tissue sections. mRNA/miRNA target pairs were identified using computational approaches and their expression was studied in purified epithelial/mesenchymal cell populations and in isolated tips and stalks from the bronchial tree. Additionally, silencing experiments in human embryonic lung mesenchymal cells and in human embryonic tip-derived lung organoids were performed, as well as organoid differentiation studies. AT2 cell markers were studied by qRT-PCR and by immunofluorescence. The TGFB-β phosphorylated pathways was analyzed with membrane protein arrays. Lung explants were cultured in air/liquid interface with/without peptides.ResultsWe identified 88 differentially expressed genes, including IGFBP3. Although IGFBP3 mRNA was detected in both epithelial and mesenchymal populations, the protein was restricted to the epithelium, indicating post-transcriptional regulation preventing IGFBP3 protein expression in the mesenchyme. MicroRNA profiling identified miR-34a as an IGFBP3 regulator. miR-34a was up-regulated in mesenchymal cells, and its silencing in human embryonic lung mesenchymal cells increased IGFBP3 levels. Additionally, IGFBP3 expression showed a marked downregulation from 7 to 12 pcw, suggesting its involvement in the differentiation process. The differentiation of human tip-derived lung embryonic organoids showed a drastic reduction in IGFBP3, supported by the scRNAseq data. IGFBP3 silencing in organoids activated an alveolar-like differentiation process characterized by stem cell markers downregulation and upregulation of AT2 markers. This process was mediated by TGFβ signalling inhibition and BMP pathway activation.ConclusionsThe IGFBP3/miR-34a axis restricts IGFBP3 expression in the embryonic undifferentiated lung epithelium, and the progressive downregulation of IGFBP3 during the pseudoglandular stage is required for alveolar differentiation.Graphical abstract

Development of the basic architecture of neocortical circuitry in the human fetus as revealed by the coupling spatiotemporal pattern of synaptogenesis along with microstructure and macroscale in vivo MR imaging.

In humans, a quantifiable number of cortical synapses appears early in fetal life. In this paper, we present a bridge across different scales of resolution and the distribution of synapses across the transient cytoarchitectonic compartments: marginal zone (MZ), cortical plate (CP), subplate (SP), and in vivo MR images. The tissue of somatosensory cortex (7-26 postconceptional weeks (PCW)) was prepared for electron microscopy, and classified synapses with a determined subpial depth were used for creating histograms matched to the histological sections immunoreacted for synaptic markers and aligned to in vivo MR images (1.5T) of corresponding fetal ages (maternal indication). Two time periods and laminar patterns of synaptogenesis were identified: an early and midfetal two-compartmental distribution (MZ and SP) and a late fetal three-compartmental distribution (CP synaptogenesis). During both periods, a voluminous, synapse-rich SP was visualized on the in vivo MR. Another novel finding concerns the phase of secondary expansion of the SP (13 PCW), where a quantifiable number of synapses appears in the upper SP. This lamina shows a T2 intermediate signal intensity below the low signal CP. In conclusion, the early fetal appearance of synapses shows early differentiation of putative genetic mechanisms underlying the synthesis, transport and assembly of synaptic proteins. "Pioneering" synapses are likely to play a morphogenetic role in constructing of fundamental circuitry architecture due to interaction between neurons. They underlie spontaneous, evoked, and resting state activity prior to ex utero experience. Synapses can also mediate genetic and environmental triggers, adversely altering the development of cortical circuitry and leading to neurodevelopmental disorders.

P-209 Development and characterization of human uterine myometrium in prenatal stage: insights from a single-cell atlas and three-dimensional imaging

Abstract Study question What’s the timepoint of human uterine myometrial formation and how dose it develop in the prenatal stage. Summary answer This study identified that the myometrium layer initially forms at postconceptional week (PCW) 11, ESR1+, IGFBP5, POSTIN may play pivotal role in myometrial development. What is known already Using immunohistochemistry staining, previous studies observed a faint expression of α-SMA in the outer mesenchyme at approximately 8-9 weeks of gestation and showed the early development of muscular layer in the uterine wall, and the expression of α-SMA becomes more prominent in the uterine corpus and cervix by 18 weeks, suggesting the maturation of smooth muscle cells may commence from 18 weeks of gestation. ESR-1 gene encodes estrogen receptor alpha, which plays a pivotal role in menstruation, pregnancy, and parturition. In adult female myometrium, estrogen performs functions in proliferation before term and contractile at term. Study design, size, duration This is an observational study. To investigate when exactly the smooth-muscle tissue appears during uterine development, we have collected 14 uterine tissue samples from 14 aborted fetuses across seven gestational time points from PCW 8 to PCW 15. Participants/materials, setting, methods Uterus from aborted fetuses from PCW8- PCW15 cover the fusion of Mullerian tube and development of Myometrium. The 10X genomics scRNA-seq and immune labeling were performed. In addition, we have integrated scRNA-seq data from adult uterine tissue for comparison. Main results and the role of chance We provide a single cell atlas of the human developing uterus, and we observed that smooth-muscle cells (SMCs), identified by ACTA2, TAGLN, MYH11, exhibited a significant increase in abundance starting from PCW 11, but weaker signals of SMCs were also detected at earlier stages. Using the iDISCO technique with continuous α-SMA labeling to examine the localization of α-SMA in the uterus between PCW10- PCW12, we observed the entire process of myometrial development in 3-D level. We utilized an oblique section of 3-D reconstruction uterus and extended it to 150 μm. Subsequently, apparent signals became visible in the middle of the uterine wall and the uterine fundus. Collectively, these findings suggest that the anatomical myometrium layer initially forms at PCW 11. We used the CytoTRACE to assess the capacity of differentiation of adult and fetal SMCs, SMCs come from fetus appeal a significant grander potential. Specifically, fetal uterine SMCs showed higher expression of ESR1, IGFBP5, and POSTN, while adult uterine SMCs exhibited higher expression of PDGFRB and IGFBP7. These results suggested ESR1+ SMCs could be the precursor of SMCs and SMCs in fetal uterus could be in a primary state. Limitations, reasons for caution While our study did not capture later stages of development or postnatal changes. Future studies are needed to understand the full spectrum of myometrial development. It is also important to note that the findings in this study are specific to human and may not directly translate to other species. Wider implications of the findings Our study provides a basis for studying abnormal myometrial development and its association with various reproductive disorders, such as the abnormal uterine development in female fetus with Turner syndrome. Potential biomarkers or therapeutic targets for uterine malformations and abnormal uterine contractility may also of research interest. Trial registration number not applicable

Developmentally dynamic changes in DNA methylation in the human pancreas

Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with > 21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome and were depleted in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying evidence for tissue-specific developmental changes in DNA methylation. This study represents the first systematic exploration of DNA methylation patterns during human fetal pancreas development and confirms the prenatal period as a time of major epigenomic plasticity.

DeepFace: Deep-learning-based framework to contextualize orofacial-cleft-related variants during human embryonic craniofacial development

Orofacial clefts (OFC) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the ‘casual’ variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson Correlation Coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50 to 0.83. Specifically, our model revealed that SNPs significantly associated with OFC tended to exhibit higher SAD scores across various variant categories compared to less-related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFC. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFC, and the model that can be extended to study other complex diseases or traits.

O13 Benchmarking a human pluripotent stem cell-derived hair-bearing skin organoid model to prenatal skin

Abstract Introduction and aims Molecular regulation of mammalian skin development has been largely derived from murine studies. The lack of human studies stems primarily from the difficulties in prenatal human skin access. There is an important need to study human prenatal skin owing to the notable differences between human and mouse skin and the paucity of mechanistic understanding of congenital skin disorders. Methods We assembled the first comprehensive multiomic reference atlas of prenatal human skin (21 samples from 7 to 16 postconception weeks), combining single-cell (235 201 cells) and spatial transcriptomic data. We followed 84 skin organoids (SkOs) using single-cell analysis over a 5-month culture period and characterized the spatial organization of the skin in mature SkOs. Prenatal skin, SkO and any relevant pre-existing single-cell RNA-Seq datasets were integrated into the analysis. Cell–cell interactions and cell-lineage determination were explored using CellPhoneDB and trajectory inferences. We evaluated the differentiation trajectory alignment between prenatal skin and SkO using the Genes2Genes analysis. Results Our findings reveal predicted factors regulating the epithelial–mesenchymal crosstalk during human hair follicle formation; progressive expression of scar-promoting genes in later gestation, potentially contributing to scarless healing properties of human prenatal skin observed during the early stages of gestation and expression of genes causing genetic skin disorders in the same cell types in prenatal human skin and SkO. We also found that SkOs morphologically recapitulate human skin development from early (periderm formation and shedding) to late (cornification and hair follicle formation) stages. We also demonstrated that SkO and prenatal skin mesenchyme differentiation into hair follicle dermal condensate and dermal papilla is highly conserved at the cellular and molecular level. Conclusions SkOs faithfully recapitulate human prenatal skin development and are a valuable model for mechanistic studies on skin development including hair follicle biology and congenital skin disorders.

Single-cell analyses reveal transient retinal progenitor cells in the ciliary margin of developing human retina

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein, we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors, which transit to late, and further to transient neurogenic progenitors, that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples, we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells, we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs, which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids.

A 3D atlas of the human developing pancreas to explore progenitor proliferation and differentiation

Aims/hypothesisRodent pancreas development has been described in great detail. On the other hand, there are still gaps in our understanding of the developmental trajectories of pancreatic cells during human ontogenesis. Here, our aim was to map the spatial and chronological dynamics of human pancreatic cell differentiation and proliferation by using 3D imaging of cleared human embryonic and fetal pancreases.MethodsWe combined tissue clearing with light-sheet fluorescence imaging in human embryonic and fetal pancreases during the first trimester of pregnancy. In addition, we validated an explant culture system enabling in vitro proliferation of pancreatic progenitors to determine the mitogenic effect of candidate molecules.ResultsWe detected the first insulin-positive cells as early as five post-conceptional weeks, two weeks earlier than previously observed. We observed few insulin-positive clusters at five post-conceptional weeks (mean ± SD 9.25±5.65) with a sharp increase to 11 post-conceptional weeks (4307±152.34). We identified a central niche as the location of onset of the earliest insulin cell production and detected extra-pancreatic loci within the adjacent developing gut. Conversely, proliferating pancreatic progenitors were located in the periphery of the epithelium, suggesting the existence of two separated pancreatic niches for differentiation and proliferation. Additionally, we observed that the proliferation ratio of progenitors ranged between 20% and 30%, while for insulin-positive cells it was 1%. We next unveiled a mitogenic effect of the platelet-derived growth factor AA isoform (PDGFAA) in progenitors acting through the pancreatic mesenchyme by increasing threefold the number of proliferating progenitors.Conclusions/interpretationThis work presents a first 3D atlas of the human developing pancreas, charting both endocrine and proliferating cells across early development.Graphical

272 Integrative Genomics Implicates Primary Impact on Cortical Development in Spina Bifida

INTRODUCTION: Spina bifida is one of the most common malformation of the central nervous system that is associated with poor neurodevelopmental outcomes. Altered neurodevelopment in spina bifida has been attributed to venting of CSF through the neural tube defect. Despite correction of the spinal defect, some patients still develop hydrocephalus with poor neurodevelopmental outcomes, suggesting incomplete understanding of disease pathogenesis. Recent human genetic efforts have identified candidate genes that confer spina bifida risk, however, the mechanisms linking gene mutations to altered neurodevelopment remain poorly understood. METHODS: We curated a list of 439 genes that confer risk to spina bifida from a whole-genome sequencing study of 310 individuals. We then mapped the spatiotemporal and cell-type specific expression of these risk genes onto bulk and single-cell RNA sequencing data of normally developing human brains covering the entire lifespan of human development. RESULTS: We found that spina bifida risk genes are enriched at post-conception weeks 5-6 and 25-38 in the developing human brain. After birth, spina bifida risk genes are enriched at postnatal months 6-10 and then after 60 years of age. At the cell type level, spina bifida risk genes are enriched in proliferative neuroprogenitor cells and microglia in the prenatal human cortex. CONCLUSIONS: Our analyses show that spina bifida risk genes are expressed in the developing human brain, suggesting a primary impact on brain development that is independent from the open caudal end of the neural tube defect. The finding that spina bifida risk genes continue to be expressed postnatally suggests a window for therapeutic intervention to optimize neurodevelopmental outcomes after birth in at least a subset of patients.

Clinical validation of the Air-Test for the non-invasive detection of perioperative atelectasis in children.

The incidence of anesthesia-induced atelectasis in children is high and closely related to episodes of hypoxemia. The Air-Test is a simple maneuver to detect lung collapse. By a step-reduction in FiO<inf>2</inf> to 0.21, a fall in pulse-oximetry hemoglobin saturation <97% unmasks the presence of collapse-related shunt in healthy lungs. The aim of this study was to validate the Air-Test as a diagnostic tool to detect perioperative atelectasis in children using lung ultrasound as a reference. We first assessed the Air-Test in a retrospective cohort of 88 anesthetized children (Retrospective study) followed by a prospective study performed in 72 children (45 postconceptional weeks to 16 years old) using a similar protocol (Validation study). We analyzed the performance of the Air-Test to detect atelectasis by an operating characteristic curve (ROC) analysis, using lung ultrasound consolidation score as reference. Preoperative SpO<inf>2</inf> was normal in both studies (retrospective 98.7±0.6%, validation 99.0±0.9%). The Air-Test, with a SpO<inf>2</inf> cut point <97%, resulted positive in 67 patients in the retrospective study (SpO<inf>2</inf> 93.3±2.1%) and in 59 in the validation study (SpO<inf>2</inf> 94.9±1.8%); both P<0.0001. In the validation study, the Air-Test showed a sensitivity of 0.91 (95% CI 0.85-0.92), specificity of 1.00 (95% CI 0.84-1) and an area under the curve (AUC) of 0.98 (95% CI 0.97-1.00). AUC between both studies was similar (P=0.16). The Air-Test is a noninvasive and accurate method to detect atelectasis in healthy anesthetized children. It can be used as a screening tool to individualize patients that can benefit from lung recruitment maneuvers.

Multiomic Analysis of Neuroinflammation and Occult Infection in Sudden Infant Death Syndrome

Antemortem infection is a risk factor for sudden infant death syndrome (SIDS)-the leading postneonatal cause of infant mortality in the developed world. Manifestations of infection and inflammation are not always apparent in clinical settings or by standard autopsy; thus, enhanced resolution approaches are needed. To ascertain whether a subset of SIDS cases is associated with neuroinflammation and occult infection. In this case-control study, postmortem fluids from SIDS cases and controls collected between July 2011 and November 2018 were screened for elevated inflammatory markers, specifically cerebrospinal fluid (CSF) neopterin and CSF and serum cytokines. CSF, liver, and brain tissue from SIDS cases with elevated CSF neopterin were subjected to metagenomic next-generation sequencing (mNGS) to probe for infectious pathogens. Brainstem tissue from a subset of these cases was analyzed by single-nucleus RNA sequencing (snRNAseq) to measure cell type-specific gene expression associated with neuroinflammation and infection. All tissue and fluid analyses were performed from April 2019 to January 2023 in a pathology research laboratory. Included was autopsy material from infants dying of SIDS and age-matched controls dying of known causes. There were no interventions or exposures. CSF neopterin levels were measured by high-performance liquid chromatography. Cytokines were measured by multiplex fluorometric assay. mNGS was performed on liver, CSF, brain, and brainstem tissue. snRNAseq was performed on brainstem tissue. A cohort of 71 SIDS cases (mean [SD] age, 55.2 [11.4] postconceptional weeks; 42 male [59.2%]) and 20 controls (mean [SD] age, 63.2 [16.9] postconceptional weeks; 11 male [55.0%]) had CSF and/or serum available. CSF neopterin was screened in 64 SIDS cases and 15 controls, with no exclusions. Tissues from 6 SIDS cases were further analyzed. For CSF neopterin measures, SIDS samples were from infants with mean (SD) age of 54.5 (11.3) postconceptional weeks (38 male [59.4%]) and control samples were from infants with mean (SD) age of 61.5 (17.4) postconceptional weeks (7 male [46.7%]). A total of 6 SIDS cases (9.3%) with high CSF neopterin were identified, suggestive of neuroinflammation. mNGS detected human parechovirus 3 (HPeV3) in tissue and CSF from 1 of these 6 cases. snRNAseq of HPeV3-positive brainstem tissue (medulla) revealed dramatic enrichment of transcripts for genes with predominately inflammatory functions compared with 3 age-matched SIDS cases with normal CSF neopterin levels. Next-generation molecular tools in autopsy tissue provide novel insight into pathogens that go unrecognized by normal autopsy methodology, including in infants dying suddenly and unexpectedly.

Spatiotemporal expression and coexpression patterns of SRPK1 in the human brain: A neurodevelopmental perspective.

SRPK1 is a splicing-related protein that plays an important role in the development and function of the human brain. This article presents evidence that SRPK1 has distinct spatiotemporal expression patterns enriched in processes related to neurodevelopmental disorders across development. We used the BrainSpan growing mammalian brain transcriptome to evaluate the distribution of SRPK1 throughout the entire brain. RNA-sequencing data were gathered from 524 tissue samples recovered from 41 postmortem brains of physiologically normal individuals spanning early developing fetus (8 postconception weeks, PCW) to later life (40 years of age). Using the Allen Human Brain Atlas (AHBA) dataset, we analyzed the spatial gene expression of 15 adult human brains. Using Toppgene, we identified genes that exhibit significant coexpression with SRPK1. We found evidence that analyzing the spatiotemporal gene expression profile and identifying coexpressed genes reveals that SRPK1 expression is involved in various neurodevelopmental and somatic events throughout the lifetime. Our findings highlight the importance of detailed maps of gene expression in the human brain for improved human-to-human translation and illustrate differences in SRPK1 expression across anatomical areas and developmental stages in healthy human brain tissue.

A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

Single-cell transcriptomic analysis of corneal organoids during development

Corneal organoids are useful tools for disease modeling and tissue transplantation; however, they have not yet been well studied during maturation. We characterized human iPSC-derived corneal organoids at 1, 2, 3, and 4months of development using single-cell RNA sequencing to determine the cellular heterogeneity at each stage. We found pluripotent cell clusters committed to epithelial cell lineage at 1month; early corneal epithelial, endothelial, and stromal cell markers at 2months; keratocytes as the largest cell population at 3months; and a large epithelial cell population at 4months. We compared organoid to fetal corneal development at different stages and found that 4-month organoids closely resemble the corneal cellular complexity of the fetal (16 post conception week) and adult cornea. Using RNA velocity trajectory analysis, we found that less differentiated cells appear to give rise to corneal epithelial cells during development.

A spatially-resolved multiomic cell atlas reveals gene regulatory networks underlying cell specification in the developing human heart

Abstract Introduction Cardiogenesis requires the differentiation and coordinated function of a large variety of cell types. Congenital heart disease (CHD), affecting 1% of live births[1], and several adult-onset heart diseases result when these processes go awry[2–4]. To understand the cellular and molecular mechanisms at play we must define the repertoire of cell types in the developing heart as well as the cellular niches within which they interact. Single-cell RNA sequencing is a powerful means of defining cell populations, however existing studies of the human foetal heart[5,6] have been hampered by low cell numbers which limits detection of rare but important cell types. To create an improved atlas we increased the number of cells, increased the age range of foetal hearts sampled, and generated spatial transcriptomic data to map the defined cells to multicellular niches of co-located and interacting cells. To better infer the transcription factors (TFs) governing cell identity using gene regulatory network (GRN) inference we also generated paired single-cell resolution RNA and chromatin accessibility profiles using Assay for Transposase-Accessible Chromatin (ATAC) sequencing. Methods We performed paired single-nuclei RNA and ATAC sequencing (10X Multiome) and single-cell RNAseq (10X 3’ RNAseq) on 23 human foetal hearts ranging from 4 to 20 post-conception weeks. In addition we generated spatial transcriptomic data (10X Visium) from these hearts. We validated cell types defined in single-cell data using protein-level immunofluorescence imaging. Results Our healthy reference atlas includes 300k cells and nuclei. Using their gene expression profile we resolved 46 fine-grained cell types (FIGURE 1). We used cell2location[7], a machine learning classifier trained on the atlas to map the defined cell types to the spatial data (FIGURE 2) and detected niches of co-locating cells, such as a macrophage population in the walls of the great vessels predicted to interact with mural cells. We were able to define location-specific transcriptional signatures such as cardiomyocytes of each chamber (as well as the compact and trabeculated myocardium) and smooth muscle cells specific to great vessels vs coronary arteries. Using a combination of RNA and ATAC data we inferred cell-specific GRNs, which implicated novel TFs such as MSX2 in sinoatrial node pacemaker cells and the enrichment of CHD genes amongst cardiomyocyte-specific GRNs. In the spatial data we showed that gene modules associated with different forms of CHD enrich in specific anatomical regions, such as the outflow tract. Discussion: Together these data constitute the most comprehensive cardiac developmental cell atlas to date. It sheds new light on the diversity of cell states, their niches, and their vulnerability to the genetic causes of CHD.Figure 1:UMAP showing atlasFigure 2:Spatial transcriptomic mapping

Comprehensive cell atlas of the first-trimester developing human brain.

The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre-oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development.