3D Genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis.

Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

The Role of Immune Factors in Shaping Fetal Neurodevelopment.

Fetal neurodevelopment in utero is profoundly shaped by both systemic maternal immunity and local processes at the maternal-fetal interface. Immune pathways are a critical participant in the normal physiology of pregnancy and perturbations of maternal immunity due to infections during this period have been increasingly linked to a diverse array of poor neurological outcomes, including diseases that manifest much later in postnatal life. While experimental models of maternal immune activation (MIA) have provided groundbreaking characterizations of the maternal pathways underlying pathogenesis, less commonly examined are the immune factors that serve pathogen-independent developmental functions in the embryo and fetus. In this review, we explore what is known about the in vivo role of immune factors in fetal neurodevelopment during normal pregnancy and provide an overview of how MIA perturbs the proper orchestration of this sequence of events. Finally, we discuss how the dysregulation of immune factors may contribute to the manifestation of a variety of neurological disorders.

The Born in Guangzhou Cohort Study enables generational genetic discoveries.

Genomic research that targets large-scale, prospective birth cohorts constitutes an essential strategy for understanding the influence of genetics and environment on human health1. Nonetheless, such studies remain scarce, particularly in Asia. Here we present the phase I genome study of the Born in Guangzhou Cohort Study2 (BIGCS), which encompasses the sequencing and analysis of 4,053 Chinese individuals, primarily composed of trios or mother-infant duos residing in South China. Our analysis reveals novel genetic variants, a high-quality reference panel, and fine-scale local genetic structure within BIGCS. Notably, we identify previously unreported East Asian-specific genetic associations with maternal total bile acid, gestational weight gain and infant cord blood traits. Additionally, we observe prevalent age-specific genetic effects on lipid levels in mothers and infants. In an exploratory intergenerational Mendelian randomization analysis, we estimate the maternal putatively causal and fetal genetic effects of seven adult phenotypes on seven fetal growth-related measurements. These findings illuminate the genetic links between maternal and early-life traits in an East Asian population and lay the groundwork for future research into the intricate interplay of genetics, intrauterine exposures and early-life experiences in shaping long-term health.

Cellular development and evolution of the mammalian cerebellum.

The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.

Single-cell atlas of the human brain vasculature across development, adulthood and disease.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Normative spatiotemporal fetal brain maturation with satisfactory development at 2 years.

Maturation of the human fetal brain should follow precisely scheduled structural growth and folding of the cerebral cortex for optimal postnatal function1. We present a normative digital atlas of fetal brain maturation based on a prospective international cohort of healthy pregnant women2, selected using World Health Organization recommendations for growth standards3. Their fetuses were accurately dated in the first trimester, with satisfactory growth and neurodevelopment from early pregnancy to 2 years of age4,5. The atlas was produced using 1,059 optimal quality, three-dimensional ultrasound brain volumes from 899 of the fetuses and an automated analysis pipeline6-8. The atlas corresponds structurally to published magnetic resonance images9, but with finer anatomical details in deep grey matter. The between-study site variability represented less than 8.0% of the total variance of all brain measures, supporting pooling data from the eight study sites to produce patterns of normative maturation. We have thereby generated an average representation of each cerebral hemisphere between 14 and 31 weeks' gestation with quantification of intracranial volume variability and growth patterns. Emergent asymmetries were detectable from as early as 14 weeks, with peak asymmetries in regions associated with language development and functional lateralization between 20 and 26 weeks' gestation. These patterns were validated in 1,487 three-dimensional brain volumes from 1,295 different fetuses in the same cohort. We provide a unique spatiotemporal benchmark of fetal brain maturation from a large cohort with normative postnatal growth and neurodevelopment.

Lineage tracing of human development through somatic mutations.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Spatiotemporal DNA methylome dynamics of the developing mouse fetus.

Cytosine DNA methylation is essential for mammalian development but understanding of its spatiotemporal distribution in the developing embryo remains limited1,2. Here, as part of the mouse Encyclopedia of DNA Elements (ENCODE) project, we profiled 168 methylomes from 12 mouse tissues or organs at 9 developmental stages from embryogenesis to adulthood. We identified 1,808,810 genomic regions that showed variations in CG methylation by comparing the methylomes of different tissues or organs from different developmental stages. These DNA elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. During late stages of fetal development, non-CG methylation accumulated within the bodies of key developmental transcription factor genes, coinciding with their transcriptional repression. Integration of genome-wide DNA methylation, histone modification and chromatin accessibility data enabled us to predict 461,141 putative developmental tissue-specific enhancers, the human orthologues of which were enriched for disease-associated genetic variants. These spatiotemporal epigenome maps provide a resource for studies of gene regulation during tissue or organ progression, and a starting point for investigating regulatory elements that are involved in human developmental disorders.

The maternal microbiome modulates fetal neurodevelopment in mice.

'Dysbiosis' of the maternal gut microbiome, in response to challenges such as infection1, altered diet2 and stress3 during pregnancy, has been increasingly associated with abnormalities in brain function and behaviour of the offspring4. However, it is unclear whether the maternal gut microbiome influences neurodevelopment during critical prenatal periods and in the absence of environmental challenges. Here we investigate how depletion and selective reconstitution of the maternal gut microbiome influences fetal neurodevelopment in mice. Embryos from antibiotic-treated and germ-free dams exhibited reduced brain expression of genes related to axonogenesis, deficient thalamocortical axons and impaired outgrowth of thalamic axons in response to cell-extrinsic factors. Gnotobiotic colonization of microbiome-depleted dams with a limited consortium of bacteria prevented abnormalities in fetal brain gene expression and thalamocortical axonogenesis. Metabolomic profiling revealed that the maternal microbiome regulates numerous small molecules in the maternal serum and the brains of fetal offspring. Select microbiota-dependent metabolites promoted axon outgrowth from fetal thalamic explants. Moreover, maternal supplementation with these metabolites abrogated deficiencies in fetal thalamocortical axons. Manipulation of the maternal microbiome and microbial metabolites during pregnancy yielded adult offspring with altered tactile sensitivity in two aversive somatosensory behavioural tasks, but no overt differences in many other sensorimotor behaviours. Together, our findings show that the maternal gut microbiome promotes fetal thalamocortical axonogenesis, probably through signalling by microbially modulated metabolites to neurons in the developing brain.

Maternal B cell signaling orchestrates fetal development in mice.

B cell participation in early embryo/fetal development and the underlying molecular pathways have not been explored. To understand whether maternal B cell absence or impaired signaling interferes with placental and fetal growth, we paired CD19-deficient (CD19-/-) mice, females with B cell-specific MyD88 (BMyD88-/-) or IL10 (BIL10-/-) deficiency as well as wild-type and MyD88-/- controls on C57Bl/6 background with BALB/c males. Pregnancies were followed by ultrasound and Doppler measurements. Implantation number was reduced in BMyD88-/- and MyD88-/- mice. Loss of MyD88 or B cell-specific deletion of MyD88 or IL10 resulted in decreased implantation areas at gestational day (gd) 5, gd8 and gd10, accompanied by reduced placental thickness, diameter and areas at gd10. Uterine artery resistance was enhanced in BIL10-/- dams at gd10. Challenge with 0.4 mg lipopolysaccharide/kg bodyweight at gd16 revealed that BMyD88-/-, BIL10-/- and CD19-/- mothers delivered preterm, whereas controls maintained their pregnancy. B cell-specific MyD88 and IL10 expression is essential for appropriate in utero development. IL10+B cells are involved in uterine blood flow regulation during pregnancy. Finally, B cell-specific CD19, MyD88 and IL10 expression influences susceptibility towards preterm birth.

Postnatal outcome of fetal cortical malformations: systematic review.

OBJECTIVE: Parental counseling for fetal malformations of cortical development (MCD) is based on data from studies in children and adults undergoing imaging investigation for abnormal neurodevelopment. However, such postnatal findings may not be applicable to prenatally diagnosed cases. The aim of this study was to review the existing data on postnatal neurodevelopmental outcome for fetuses diagnosed with MCD. METHODS: A literature search was conducted in PubMed, Web of Science and EMBASE for articles published between 2013 and 2023, using standardized keywords to describe fetal cortical malformations. Full-text articles were accessed for the retrieved citations and data on participant characteristics, imaging findings, and pregnancy and neonatal outcomes were extracted. Fetal MCD was defined as either complex or isolated, according to the presence or absence, respectively, of additional brain or extracranial defects. RESULTS: Overall, 30 articles including 371 cases of fetal MCD were reviewed. The cases were classified as complex (n = 324), isolated (n = 21) or unknown (n = 26). There were 144 terminations and four stillbirths, with pregnancy outcome unreported in 149 cases. A total of 108 cases had postnatal magnetic resonance imaging or postmortem examination data available. In nine of these cases, a diagnosis of complex fetal MCD was changed to isolated MCD after birth, and one case was found not to have MCD. There were 74 live births, for which postnatal neurodevelopment data were available in only 30 cases. Normal neurodevelopmental outcome was reported in seven (23.3% (95% CI, 9.9-42.2%)) infants, with the remaining 23 exhibiting various levels of neurodevelopmental delay (three mild, seven moderate and 13 severe) from 6 months to 7 years of age. CONCLUSIONS: Most reviewed cases of fetal MCD were complex in nature and underwent termination of pregnancy. There is a paucity of data on postnatal neurological development in fetuses diagnosed with MCD. The available data suggest antenatal overdiagnosis of case severity in about 5% of cases with known outcome, and either normal neurodevelopment or mild neurodevelopmental delay in approximately one-third of liveborn cases with neurological follow-up. © 2024 The Author(s). Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Whole-body non-forensic fetal virtopsy using postmortem magnetic resonance imaging at 7 Tesla vs classical autopsy.

OBJECTIVE: To determine the diagnostic accuracy of virtual autopsy using whole-body postmortem ultra-high field magnetic resonance imaging (MRI) at 7 Tesla (T), using a short T2-weighted imaging (T2-WI) protocol, compared with classical autopsy, for detecting structural abnormalities in small second-trimester fetuses. METHODS: Thirty consecutive fetuses at 13-19 weeks' gestation (weight, 17-364 g) were included following spontaneous pregnancy loss or termination of pregnancy. After fixation in 10% formaldehyde solution (48 h to 1 week), all fetuses were scanned using a two-dimensional turbo high-resolution T2-WI protocol with multislice relaxation time, followed by an invasive autopsy. The diagnostic accuracy of virtual autopsy vs classical autopsy was calculated for 990 anatomical structures (30 fetuses × 33 items). Sensitivity, specificity, positive and negative predictive values and Cohen's κ coefficient of agreement, with their 95% CIs, as well as the McNemar test, were used to evaluate the accuracy and agreement of the two diagnostic methods. Analysis was stratified by anatomical segment (nervous, pulmonary, cardiovascular, digestive, renal, facial and skeletal) and across three gestational-age intervals (13-14, 15-16 and 17-19 weeks). RESULTS: Considering classical autopsy as the gold standard, virtual autopsy had a sensitivity of 92.04% (95% CI, 85.42-96.29%) and a specificity of 97.87% (95% CI, 94.64-99.42%), with a positive predictive value of 96.30% (95% CI, 90.78-98.56%) and a negative predictive value of 95.34% (95% CI, 91.61-97.45%), achieving a diagnostic accuracy of 95.68% (95% CI, 92.73-97.68%) for detecting structural abnormalities in second-trimester fetuses. Cohen's κ for virtual vs classical autopsy was 0.907. The diagnostic ability of virtual autopsy at 7 T for malformed fetuses was superior to that of classical autopsy for analyzing the nervous system in small fetuses with pronounced autolysis, equivalent to that of classical autopsy when analyzing pulmonary, cardiovascular and renal systems and inferior when evaluating the fetal intestines. The sensitivity of virtual autopsy at 7 T for describing structural abnormalities increased with gestational age. CONCLUSION: Virtual fetal autopsy using 7-T MRI and a turbo high-resolution T2-WI protocol with multislice relaxation time is a feasible postmortem diagnostic tool for the identification of fetal structural anomalies. © 2024 The Author(s). Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Early role for a Na+,K+-ATPase (ATP1A3) in brain development.

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function catalyzed by the Na+,K+-ATPase α-subunit. Here, we describe ATP1A3 variants encoding dysfunctional α3-subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we built an ATP1A3 transcriptional atlas of fetal cortical development using mRNA in situ hybridization and transcriptomic profiling of ∼125,000 individual cells with single-cell RNA sequencing (Drop-seq) from 11 areas of the midgestational human neocortex. We found that fetal expression of ATP1A3 is most abundant to a subset of excitatory neurons carrying transcriptional signatures of the developing subplate, yet also maintains expression in nonneuronal cell populations. Moving forward a year in human development, we profiled ∼52,000 nuclei from four areas of an infant neocortex and show that ATP1A3 expression persists throughout early postnatal development, most predominantly in inhibitory neurons, including parvalbumin interneurons in the frontal cortex. Finally, we discovered the heteromeric Na+,K+-ATPase pump complex may form nonredundant cell-type-specific α-ß isoform combinations, including α3-ß1 in excitatory neurons and α3-ß2 in inhibitory neurons. Together, the developmental malformation phenotype of affected individuals and single-cell ATP1A3 expression patterns point to a key role for α3 in human cortex development, as well as a cell-type basis for pre- and postnatal ATP1A3-associated diseases.

Development of nerves and vessels in the penis during the human fetal period.

INTRODUCTION: Although nerves and vessels of the penis play important role in erection, there are few studies on their development in human fetus. Therefore, the objective of the present study is to analyze, quantitatively, in the corpora cavernosa and corpus spongiosum, the development of the nerves and vessels in the fetal penis at different gestational ages. MATERIAL AND METHODS: Fifty-six fresh, macroscopically normal human fetuses aged from 13 to 36 weeks post-conception (WPC) were used. Gestational age was determined by the foot length criterion. Penises were immediately fixed in 10% formalin, and routinely processed for paraffin embedding, after which tissue sections from the mid-shaft were obtained. We used immunohistochemical staining to analyze the nerves and vessels in the corpus cavernous and in the corpus spongiosum. These elements were identified and quantified as percentage by using the Image-J software. RESULTS: The quantitative analysis showed that the percentage of nerves varied from 3.03% to 20.35% in the corpora cavernosa and from 1.89% to 23.88% in the corpus spongiosum. The linear regression analysis indicated that nerves growth (incidence) in the corpora cavernosa and corpus spongiosum correlated significantly and positively with fetal age (r2=0.9421, p<0.0001) and (r2=0.9312, p<0.0001), respectively, during the whole fetal period studied. Also, the quantitative analysis showed that the percentage of vessels varies from 2.96% to 12.86% in the corpora cavernosa and from 3.62% to 14.85% in the corpus spongiosum. The linear regression analysis indicated that vessels growth (appearance) in the corpora cavernosa and corpus spongiosum correlated significantly and positively with fetal age (r2=0.8722, p<0.0001) and (r2=0.8218, p<0.0001), respectively, during the whole fetal period studied. In addition, the linear regression analysis demonstrated a more intense growth rate of nerves in the corpus spongiosum during the 2nd trimester of gestation, when compared with nerves in the corpora cavernosa. In addition, the linear regression analysis demonstrated a more intense growth rate of vessels in the corpus spongiosum when compared with the corpora cavernosa, during the whole fetal period studied. CONCLUSIONS: In the fetal period, the human penis undergoes major developmental changes, notably in the content and distribution of nerves and vessels. We found strong correlation between nerves and vessels growth (amount) with fetal age, both in the corpora cavernosa and corpus spongiosum. There is significant greater proportional number of nerves than vessels during the whole fetal period studied. Also, nerves and vessels grow in a more intense rate than that of the corpora cavernosa and corpus spongiosum areas.

A mutation affecting laminin alpha 5 polymerisation gives rise to a syndromic developmental disorder.

Laminin alpha 5 (LAMA5) is a member of a large family of proteins that trimerise and then polymerise to form a central component of all basement membranes. Consequently, the protein plays an instrumental role in shaping the normal development of the kidney, skin, neural tube, lung and limb, and many other organs and tissues. Pathogenic mutations in some laminins have been shown to cause a range of largely syndromic conditions affecting the competency of the basement membranes to which they contribute. We report the identification of a mutation in the polymerisation domain of LAMA5 in a patient with a complex syndromic disease characterised by defects in kidney, craniofacial and limb development, and by a range of other congenital defects. Using CRISPR-generated mouse models and biochemical assays, we demonstrate the pathogenicity of this variant, showing that the change results in a failure of the polymerisation of α/ß/γ laminin trimers. Comparing these in vivo phenotypes with those apparent upon gene deletion in mice provides insights into the specific functional importance of laminin polymerisation during development and tissue homeostasis.