Divergent Lineages Research Articles

Atlas of multilineage stem cell differentiation reveals TMEM88 as a developmental regulator of blood pressure.

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across developmental lineages. Here, we engineer barcoded induced pluripotent stem cells to generate an atlas of multilineage differentiation from pluripotency, encompassing an eight-day time course with modulation of WNT, BMP, and VEGF signalling pathways. Annotation of in vitro cell types with reference to in vivo development reveals diverse mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Interrogation of temporal and signalling-specific gene expression in this atlas, evaluated against cell type-specific gene expression in human complex trait data highlights the WNT-inhibitor gene TMEM88 as a regulator of mesendodermal lineages influencing cardiovascular and anthropometric traits. Genetic TMEM88 loss of function models show impaired differentiation of endodermal and mesodermal derivatives in vitro and dysregulated arterial blood pressure in vivo. Together, this study provides an atlas of multilineage stem cell differentiation and analysis pipelines to dissect genetic determinants of mammalian developmental physiology.

Footprints of Human Migration in the Population Structure of Wild Baker's Yeast.

Humans have a long history of fermenting food and beverages that led to domestication of the baker's yeast, Saccharomyces cerevisiae. Despite their tight companionship with humans, yeast species that are domesticated or pathogenic can also live on trees. Here we used over 300 genomes of S. cerevisiae from oaks and other trees to determine whether tree-associated populations are genetically distinct from domesticated lineages and estimate the timing of forest lineage divergence. We found populations on trees are highly structured within Europe, Japan, and North America. Approximate estimates of when forest lineages diverged out of Asia and into North America and Europe coincide with the end of the last ice age, the spread of agriculture, and the onset of fermentation by humans. It appears that migration from human-associated environments to trees is ongoing. Indeed, patterns of ancestry in the genomes of three recent migrants from the trees of North America to Europe could be explained by the human response to the Great French Wine Blight. Our results suggest that human-assisted migration affects forest populations, albeit rarely. Such migration events may even have shaped the global distribution of S. cerevisiae. Given the potential for lasting impacts due to yeast migration between human and natural environments, it seems important to understand the evolution of human commensals and pathogens in wild niches.

Phylogenetics and Evolutionary Dynamics of Yunnan Acrididae Grasshoppers Inferred from 17 New Mitochondrial Genomes

Yunnan Province, a global biodiversity hotspot, hosts a diverse array of Acrididae grasshoppers essential for ecosystem dynamics and agriculture. To elucidate the phylogenetic relationships and evolutionary history of this group, we sequenced and analyzed complete mitochondrial genomes from 17 Acrididae species endemic to Yunnan, with genome lengths ranging from 15,403 to 15,943 base pairs. These data were integrated with mitochondrial sequences from 46 additional species to construct comprehensive phylogenetic trees. The maximum-likelihood tree identified four major clades with robust support (bootstrap values > 90%), revealing significant lineage diversification during the Early Eocene (51.94 million years ago, Mya) and subsequent radiations in the Miocene (~20 Mya) and Pliocene (~5 Mya). These divergence times correlate with major geological events and climatic shifts in the region, such as the uplift of the Tibetan Plateau and the intensification of the Asian monsoon. Notably, several species within the Coptacrinae and Oxyinae subfamilies, particularly Pseudodotraulia cornuata and Spathosternum prasiniferum, were found to be polyphyletic, indicating the necessity for taxonomic revisions. Further cluster analyses of codon usage bias and genetic distance support these taxonomic revisions within Acrididae. This robust phylogenetic framework underpins conservation strategies aimed at preserving Yunnan’s rich grasshopper biodiversity and informs updates to their phylogeny.

And growth on form? How tissue expansion generates novel shapes, colours and enhance biological functions of Turing colour patterns of Eukaryotes.

Evidenced in zebrafishes skin and Mimulus petal, Turing-like mechanisms are probably responsible for many periodic color patterns of Eukaryotes. They are characterized by the mathematical relationships linking their cellular or molecular actors, the periodicity and the geometrical range of the patterns they produce: spots, stripes or mazes. Nevertheless, some periodic patterns such as leopard iconic rosettes required additional ingredients to explain their formation. Growth being the main candidate, we extensively explore its multiple facets, at the Eukaryotes scale. We show that far beyond the particular feline coat pattern, putative-growth Turing color patterns are present in many diverse lineages of plants and animals and seem absent in Fungi and unicellular lineages. Using models, we show the many ways growth can induce new shapes and colors, and that putative-growth pattern locations correlates with tissue hot spots of growth, suggesting the latter as the underlying mechanism. By reverse reasoning, we show that growth effects could reveal crucial information about pattern formation. We show how putative growth patterns can contribute to influence organisms visibility, thereby improving camouflage or aposematism. Our results demonstrate the range of morphogenetic roles that tissue expansion can take, by interacting with a scale-sensitive mechanism, here Turing-like patterning. Considering this extensive overview of its biological importance, both qualitatively and quantitatively, links between growth and form might more than ever needed to be explored.

A Genome-Wide Analysis of Nuclear Mitochondrial DNA Sequences (NUMTs) in Chrysomelidae Species (Coleoptera)

Nuclear mitochondrial DNA sequences (NUMTs) are mitochondrial DNA fragments in the nuclear genome, and their unclear distribution in Chrysomelidae species hinders the selection of accurate molecular markers for species identification and phylogenetic analysis. Our study presents a genome-wide survey of NUMTs in 32 Chrysomelidae species. Filtering strategies based on sequence length and open reading frame (ORF) features were employed to identify mitochondrial protein-coding genes (PCGs) minimally affected by NUMTs. Phylogenetic relationships were inferred from both mitochondrial PCG datasets and a COX1 dataset containing NUMTs. Our results show that NUMTs are chromosomally specific, species-specific, and widely distributed. ATP8, COX1, ND1, and ND4 are identified as relatively reliable molecular markers. Phylogenetic analysis is influenced by NUMTs and other factors such as sequence type and saturation. A total of 66 independent COX1 gene nuclear integration events were estimated across 32 species, mostly from distinct mitochondrial lineages. These findings suggest that NUMTs reflect key evolutionary processes such as gene flow and mitochondrial lineage diversification. Their prevalence emphasizes the need for refined molecular markers in species identification and phylogenetic analysis, while also highlighting the importance of NUMTs in understanding mitochondrial DNA integration and their contribution to species’ evolutionary history.

Inferring cell trajectories of spatial transcriptomics via optimal transport analysis.

The integration of cell transcriptomics and spatial position to organize differentiation trajectories remains a challenge. Here, we introduce SpaTrack, which leverages optimal transport to reconcile both gene expression and spatial position from spatial transcriptomics into the transition costs, thereby reconstructing cell differentiation. SpaTrack can construct detailed spatial trajectories that reflect the differentiation topology and trace cell dynamics across multiple samples over temporal intervals. To capture the dynamic drivers of differentiation, SpaTrack models cell fate as a function of expression profiles influenced by transcription factors over time. By applying SpaTrack, we successfully disentangle spatiotemporal trajectories of axolotl telencephalon regeneration and mouse midbrain development. Diverse malignant lineages expanding within a primary tumor are uncovered. One lineage, characterized by upregulated epithelial mesenchymal transition, implants at the metastatic site and subsequently colonizes to form a secondary tumor. Overall, SpaTrack efficiently advances trajectory inference from spatial transcriptomics, providing valuable insights into differentiation processes.

Cephalopod sex determination and its ancient evolutionary origin.

Octopuses, squids, and cuttlefishes-the coleoid cephalopods-are a remarkable branch in the tree of life whose members exhibit a repertoire of sophisticated behaviors.1 As a clade, coleoids harbor an incredible variety of novel traits, including the most complex nervous system among invertebrates, derived camera-type eyes, and rapid adaptive camouflage abilities.2,3 The burst of evolutionary novelty that distinguishes cephalopods is even more striking in a phylogenetic context; cephalopods are a deeply diverged lineage that last shared a common ancestor with other extant molluscs in the Cambrian period, roughly 550 million years ago.4,5 With recent advances in genome sequencing technologies, we have the capability to explore the genomic foundations of cephalopod novelties. Here, using PacBio long-read sequencing of genomic DNA and Iso-Seq full-length mRNA sequencing, we provide a novel chromosome-scale reference genome and annotation for a female California two-spot octopus (O.bimaculoides). Our assembly reveals that the female octopus has just one sex chromosome, consistent with a ZO karyotype, whereas the male has two (ZZ), providing the first evidence of genetic sex determination in cephalopods. We use our assembly and annotation in combination with existing genomic information from other cephalopods to create the first whole-genome alignments from this group and demonstrate that the sex chromosome is of an ancient origin, before the radiation of extant cephalopods approximately 480 million years ago,4 and has been conserved to the present day in all cephalopod genomes available.

Lineage Diversification and Population Dynamics of the Qinghai Toad-Headed Agama (Phrynocephalus vlangalii) on the Qinghai–Tibet Plateau, with Particular Attention to the Northern Slope of the Kunlun–Arjin Mountains

Lineage Diversification and Population Dynamics of the Qinghai Toad-Headed Agama (Phrynocephalus vlangalii) on the Qinghai–Tibet Plateau, with Particular Attention to the Northern Slope of the Kunlun–Arjin Mountains

Comparative population genomics unveils congruent secondary suture zone in Southwest Pacific Hydrothermal Vents.

How the interplay of biotic and abiotic factors shapes current genetic diversity at the community level remains an open question, particularly in the deep sea. Comparative phylogeography of multiple species can reveal the influence of past climatic events, geographic barriers, and species life history traits on spatial patterns of genetic structure across lineages. To shed light on the factors that shape community-level genetic variation and to improve our understanding of deep-sea biogeographic patterns, we conducted a comparative population genomics study on seven hydrothermal vent species co-distributed in the Back-Arc Basins (BABs) of the Southwest Pacific region. Using ddRAD-seq, we compared the range-wide distribution of genomic diversity across species and discovered a shared phylogeographic break. Demogenetic inference revealed shared histories of lineage divergence and a secondary contact. Low levels of asymmetric gene flow probably occurred in most species between the Woodlark and North Fiji basins, but the exact location of contact zones varied from species to species. For two species, we found individuals from the two lineages co-occurring in sympatry in Woodlark Basin. Although species exhibit congruent patterns of spatial structure (Eastern vs Western sites), they also show variation in the degree of divergence among lineages across the suture zone. Our results also show heterogeneous gene flow across the genome, indicating possible partial reproductive isolation between lineages and early speciation. Our comparative study highlights the pivotal role of historical and contemporary factors, underscoring the need for a comprehensive approach-especially in addressing knowledge gaps on the life history traits of deep-sea species.

P-1365. Cefazolin Inoculum Effect in Methicillin Susceptible Staphylococcus aureus Recovered From the Nasal Cavity of Patients in Intensive Care Units in Colombia

Abstract Background Cefazolin is an alternative for the treatment of MSSA infections and first choice option for surgical prophylaxis. The cefazolin inoculum effect (CzIE), the increase in the MIC at high bacterial inocula is associated to therapeutic failures and increased mortality in deep-seated MSSA infections. The prevalence of the CzIE among nasal colonizing MSSA from ICU patients is currently unknown. The aim of this study was to investigate the CzIE in nasal MSSA recovered from patients admitted to ICUs in Colombia. Methods We evaluated 33 MSSA isolates recovered from nasal swabs of 29 patients admitted to ICU at 6 high-complexity Colombian hospitals (2019-2023). CFZ MIC using gold standard broth microdilution at high inoculum was done. The modified nitrocefin-based rapid test to identify the CzIE was performed, and diagnostic performance metrics were calculated. Whole genome sequencing was used to characterize BlaZ types and allotypes, Clonal Complexes CC and Accessory Gene Regulator (Agr) types. Results The CzIE was identified in 54.5% of nasal MSSA isolates. A high prevalence of nasal colonization with MSSA exhibiting the CzIE was detected (59%, 17 patients). Among 18 MSSA with the CzIE, type A BlaZ was the predominant β-lactamase (61%) and BlaZ-2 was the most common allotype (50%). Whereas among 15 MSSA lacking the CzIE, BlaZ type C was the most frequent (47%) and BlaZ-1 was the allotype predominant (40%). A high diversity of genetic lineages with eight CC was detected among the 33 MSSA. CC30 was the predominant lineage in MSSA displaying the CzIE (44%), while CC5 and CC30 (27% each one) were the most common in MSSA without the CzIE. Agr-III and Agr-I were predominant Agr types in 56% and 47% of MSSA with the CzIE and lacking the CzIE, respectively. Compared to the gold standard the modified rapid test identified nasal MSSA isolates showing the CzIE with a sensitivity of 100%, specificity of 93% and an overall accuracy of 97%. Conclusion We found an unexpected high prevalence (59%) of ICU patients colonized by MSSA exhibiting the CzIE, showing similar genetic features to MSSA with CzIE from invasive infections. Furthermore, our findings suggest that the nasal cavity of ICU patients may represent a reservoir for the dissemination of MSSA showing the CzIE, highlighting the importance of screening and surveillance. Disclosures All Authors: No reported disclosures

Climatically Specialized Lineages of Batrachochytrium dendrobatidis, and its Likely Asian Origins.

Chytridiomycosis is a wildlife disease that has caused significant declines in amphibian populations and species extinctions worldwide. Asia, where the causal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamndrivorans (Bsal) originated, has not witnessed mass die-offs. It is hypothesized that Asian amphibians may have evolved immunity to clinical Batrachochytrium infection, but this has not been explored in depth due to limited knowledge of endemic lineages and infection patterns. We investigated Bd's genetic diversity and infection patterns in south China's Guangxi region using the internal transcribed spacer (ITS) marker and nested PCR. Across the 17 forest sites studied (N = 1088 individuals; 1012 adults and 76 tadpoles), the overall prevalence of Bd infection was 4.74% in adult individuals and 5.26% in tadpoles. We found seven new haplotypes, four of which were closely related to the BdASIA-1 lineage from South Korea. The most prevalent haplotype (genetically similar to BdASIA-3) was found in 11 out of 15 infected species, including a salamander with non-lethal skin lesions. A generalized linear model of our environmental data indicates that Bd infection is correlated with mean temperature of the warmest quarter and elevation, with higher infection prevalence associated with lower temperature and relatively higher elevation in southern China. Our findings suggest significant undiscovered genetic diversity of Asian Bd lineages in this region. Longer-term studies are required to further investigate Bd diversity, prevalence, seasonality, and impact on native species and populations in Southern China and across the region of origin in Asia.

YHSeqY3000 panel captures all founding lineages in the Chinese paternal genomic diversity database

BackgroundThe advancements in second-/third-generation sequencing technologies, alongside computational innovations, have significantly enhanced our understanding of the genomic structure of Y-chromosomes and their unique phylogenetic characteristics. These researches, despite the challenges posed by the lack of population-scale genomic databases, have the potential to revolutionize our approach to high-resolution, population-specific Y-chromosome panels and databases for anthropological and forensic applications.ObjectivesThis study aimed to develop the highest-resolution Y-targeted sequencing panel, utilizing time-stamped, core phylogenetic informative mutations identified from high-coverage sequences in the YanHuang cohort. This panel is intended to provide a new tool for forensic complex pedigree search and paternal biogeographical ancestry inference, as well as explore the general patterns of the fine-scale paternal evolutionary history of ethnolinguistically diverse Chinese populations.ResultsThe sequencing performance of the East Asian-specific Y-chromosomal panel, including 2999-core SNP variants, was found to be robust and reliable. The YHSeqY3000 panel was designed to capture the genetic diversity of Chinese paternal lineages from 3500 years ago, identifying 408 terminal lineages in 2097 individuals across 41 genetically and geographically distinct populations. We identified a fine-scale paternal substructure that was correlating with ancient population migrations and expansions. New evidence was provided for extensive gene flow events between minority ethnic groups and Han Chinese people, based on the integrative Chinese Paternal Genomic Diversity Database.ConclusionsThis work successfully integrated Y-chromosome-related basic genomic science with forensic and anthropological translational applications, emphasizing the necessity of comprehensively characterizing Y-chromosome genomic diversity from genomically under-representative populations. This is particularly important in the second phase of our population-specific medical or anthropological genomic cohorts, where dense sampling strategies are employed.

Rethinking large scale phylogenomics with EukPhylo v1.0, a flexible toolkit to enable phylogeny-informed data curation and analyses of diverse eukaryotic lineages.

Eukaryotic diversity is largely microbial, with macroscopic lineages (plant, animals and fungi) nesting among a plethora of diverse protists. Understanding the evolutionary relationships among eukaryotes is rapidly advancing through omics analyses, but phylogenomics are challenging for microeukaryotes, particularly uncultivable lineages, as single-cell sequencing approaches generate a mixture of sequences from hosts, associated microbiomes, and contaminants. Moreover, many analyses of eukaryotic gene families and phylogenies rely on boutique datasets and methods that are challenging for other research groups to replicate. To address these challenges, we present EukPhylo v1.0, a modular, user-friendly pipeline that enables effective data curation through phylogeny-informed contamination removal, estimation of homologous gene families (GFs), and generation of both multisequence alignments and gene trees. Analyses can use a hook database of ~15k ancient GFs or users can easily replace this hook with a set of gene families of interest. We demonstrate the power of EukPhylo, including a suite of stand-alone utilities, through analyses of 500 conserved GFs sampled from 1,000 diverse species of eukaryotes, bacteria and archaea. We show improvements in estimates of the eukaryotic tree of life, recovering clades that are well established in the literature, through successive rounds of curation using the EukPhylo contamination loop. The final trees corroborate numerous hypotheses in the literature (e.g. Opisthokonta, Rhizaria, Amoebozoa) while challenging others (e.g. CRuMs, Obazoa, Diaphoretickes). We believe that the flexibility and transparency of EukPhylo sets standards for curation of omics data for future studies.

DNA barcode and sequence related amplified polymorphism markers reveal phylogenetic relationship among Pinus species representing closed lineage divergence

DNA barcode and sequence related amplified polymorphism markers reveal phylogenetic relationship among Pinus species representing closed lineage divergence

Exploring Environmental Microfungal Diversity Through Serial Single Cell Screening.

Known for its remarkable diversity and ecological importance, the fungal kingdom remains largely unexplored. In fact, the number of unknown and undescribed fungi is predicted to exceed the number of known fungal species by far. Despite efforts to uncover these dark fungal taxa, we still face inherent sampling biases and methodological limitations. Here, we present a framework that combines taxonomic knowledge, molecular biology and data processing to explore the fungal biodiversity of enigmatic aquatic fungal lineages. Our work is based on serial screening of environmental fungal cells to approach unknown fungal taxa. Microscopic documentation is followed by DNA analysis of laser micro-dissected cells, coupled with a ribosomal operon barcoding step realised by long-read sequencing, followed by an optional whole genome sequencing step. We tested this approach on a range of aquatic fungal cells mostly belonging to the ecological group of aquatic hyphomycetes derived from environmental samples. From this initial screening, we were able to identify 60 potentially new fungal taxa in the target dataset. By extending this methodology to other fungal lineages associated with different habitats, we expect to increasingly characterise the molecular barcodes of dark fungal taxa in diverse environmental samples. This work offers a promising solution to the challenges posed by unknown and unculturable fungi and holds the potential to be applied to the diverse lineages of undescribed microeukaryotes.

Genomic Rewilding of Domestic Animals: The Role of Hybridization and Selection in Wolfdog Breeds.

Background/Objectives: The domestication of the grey wolf (Canis lupus) and subsequent creation of modern dog breeds have significantly shaped the genetic landscape of domestic canines. This study investigates the genomic effects of hybridization and breeding management practices in two hybrid wolfdog breeds: the Czechoslovakian Wolfdog (CSW) and the Saarloos Wolfdog (SAW). Methods: We analyzed the genomes of 46 CSWs and 20 SAWs, comparing them to 12 German Shepherds (GSHs) and 20 wolves (WLFs), which served as their ancestral populations approximately 70-90 years ago. Results: Our findings highlight that hybridization can increase genetic variability and mitigate the effects of inbreeding, as evidenced by the observed heterozygosity levels in both wolfdog breeds. However, the SAW genome revealed a higher coefficient of inbreeding and longer runs of homozygosity compared to the CSW, reflecting significant inbreeding during its development. Discriminant Analysis of Principal Components and fixation index analyses demonstrate that the CSW exhibits closer genetic proximity to the GSH than the SAW, likely due to differences in the numbers of GSHs used during their creation. Maximum likelihood clustering further confirmed clear genetic differentiation between these hybrid breeds and their respective ancestors, while shared ancestral polymorphism was detectable in all populations. Conclusions: These results highlight the role of controlled hybridization with captive-bred wolves and peculiar breeding strategies in shaping the genetic structure of wolfdog breeds. To ensure the long-term genetic health of these breeds, it is recommended to promote adequate and sustainable breeding practices to maintain genetic diversity, minimize inbreeding, and incorporate the careful selection of unrelated individuals from diverse lineages, while avoiding additional, uncontrolled crossings with wild wolves.

Rapid radiation of a plant lineage sheds light on the assembly of dry valley biomes.

Southwest China is characterized by high plateaus, large mountain systems, and deeply incised dry valleys formed by major rivers and their tributaries. Despite the considerable attention given to alpine plant radiations in this region, the timing and mode of diversification of the numerous dry valley plant lineages remain unknown. To address this knowledge gap, we investigated the macroevolution of Isodon (Lamiaceae), a lineage commonly distributed in the dry valleys in southwest China and wetter areas of Asia and Africa. We reconstructed a robust phylogeny encompassing nearly 90% of the approximately 140 extant Isodon species using transcriptome and genome-resequencing data. Our results suggest a rapid radiation of Isodon during the Pliocene that coincided with a habit shift from herbs to shrubs and a habitat shift from humid areas to dry valleys. The shrubby growth form likely acted as a preadaptation allowing for the movement of Isodon species into these dry valleys. Ecological analyses highlight drought-related factors as key drivers influencing the niche preferences of different growth forms and species richness of Isodon. The interplay between topography and the development of the East Asian monsoon since the middle Miocene likely contributed to the formation of the dry valley biome in southwest China. This study enhances our understanding of evolutionary dynamics and ecological drivers shaping the distinctive flora of southwest China and reveals the strategies employed by montane plants in response to climate change and dryland expansion, thus facilitating conservation efforts globally.

Nr4a1 and Nr4a3 redundantly control clonal deletion and contribute to an anergy-like transcriptome in auto-reactive thymocytes to impose tolerance in mice

The Nr4a nuclear hormone receptors are transcriptionally upregulated in response to antigen recognition by the T cell receptor (TCR) in the thymus and are implicated in clonal deletion, but the mechanisms by which they operate are not clear. Moreover, their role in central tolerance is obscured by redundancy among the Nr4a family members and by their reported functions in Treg generation and maintenance. Here we take advantage of competitive bone marrow chimeras and the OT-II/RIPmOVA model to show that Nr4a1 and Nr4a3 are essential for the upregulation of Bcl2l11/BIM and thymic clonal deletion by self-antigen. Importantly, thymocytes lacking Nr4a1/3 acquire an anergy-like signature after escaping clonal deletion and Treg lineage diversion. We further show that the Nr4a family helps mediate a broad transcriptional program in self-reactive thymocytes that resembles anergy and may operate at the margins of canonical thymic tolerance mechanisms to restrain self-reactive T cells after thymic egress.

Cryo-EM structure and evolutionary history of the conjugation surface exclusion protein TraT

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF reveal that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol and palmitic acid modified α-helical barrel domain. Unexpectedly, we identify chromosomal TraT homologues from multiple Gram-negative phyla which form numerous divergent lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences are found in multiple distinct lineages, including two separate clades incorporating TraT from Enterobacteriaceae IncF/F-like and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions.

Spatial transcriptomic characterization of a Carnegie stage 7 human embryo.

Gastrulation marks a pivotal stage in mammalian embryonic development, establishing the three germ layers and body axis through lineage diversification and morphogenetic movements. However, studying human gastrulating embryos is challenging due to limited access to early tissues. Here we show the use of spatial transcriptomics to analyse a fully intact Carnegie stage 7 human embryo at single-cell resolution, along with immunofluorescence validations in a second embryo. Employing 82 serial cryosections and Stereo-seq technology, we reconstructed a three-dimensional model of the embryo. Our findings reveal early specification of distinct mesoderm subtypes and the presence of the anterior visceral endoderm. Notably, primordial germ cells were located in the connecting stalk, and haematopoietic stem cell-independent haematopoiesis was observed in the yolk sac. This study advances our understanding of human gastrulation and provides a valuable dataset for future research in early human development.