Xuefei Yuan

Understanding the genetic changes that drove the evolution of human-specific traits is a central goal in biology. Changes in gene expression are thought to be a major source of phenotypic innovations ^1,2 , but identifying the underlying changes in cis-regulatory elements (CREs) along the human lineage has been difficult: functional data from great apes are scarce and unobtainable for extinct hominins, and pinpointing causal substitutions requires knowing which sequences determine CRE activity. Here, we used AlphaGenome ³ , a sequence-to-function deep-learning model, to predict the cis-regulatory effects of ∼17 million single-nucleotide substitutions specific to the human lineage and traced their consequences for gene expression evolution across 305 tissues and cell types. The 71,345 predicted cis-regulatory gains lie mostly outside human accelerated regions ^4,5 and arose either through creation of new CREs or, more often, through co-option of CREs ancestrally accessible in other tissues. They are also enriched for distal enhancers and for target gene functions characteristic of the corresponding tissues, suggesting that they contributed to the evolution of tissue-specific biology in humans. Genes linked to more gains were more likely to be upregulated in humans, and in silico reversion to ancestral alleles traced such upregulations to individual substitutions creating transcription factor binding motifs. Comparison with Neandertal and Denisovan genomes revealed that nearly all gains now fixed in humans predate the split from archaic hominins, yet a few modern-human-specific gains contribute substantially to gene upregulation. Our study provides a genome-wide, tissue-resolved map of cis-regulatory changes and their contribution to gene expression evolution along the human lineage.

The liver is a central organ with essential roles in processes such as nutritional metabolism, detoxification, and immune defense ^1–6 . It has been instrumental in the adaptation of mammalian species to diverse environments, as reflected by its rapid molecular evolution ^7,8 . However, the origins and evolutionary dynamics of liver cell types and their structural organization remain largely unexplored. Here we report evolutionary analyses of transcriptome and chromatin accessibility data for liver cells from 17 species, spanning all major feeding strategies (herbivory, omnivory, carnivory, insectivory), great apes (including humans), placental clades (Afrotheria, Xenarthra, Laurasiatheria, Euarchontoglires), major mammalian lineages (placentals, marsupials, monotremes), and a bird as outgroup. Integrated with spatial transcriptomics, our data reveal that liver zonation—the compartmentalization of hepatocyte functions along the lobule, the liver’s fundamental anatomical and functional unit—is conserved across mammals but absent in other vertebrates. We find that zonation originated in the mammalian ancestor, driven by the emergence of WNT and R-spondin signaling from central vein endothelial cells, which activate central hepatocyte gene expression via the transcription factor TCF7L2. Despite this conserved architecture and signaling, genes with zonated expression exhibit rapid evolutionary turnover. Consistently, hepatocytes evolve fast, likely due to reduced selective constraints, enabling adaptive changes under positive selection. Alongside immune cells, hepatocytes are therefore key drivers of the liver’s rapid evolution and functional innovations. In great apes, we identify human-specific shifts in zonation and cell-type-specific expression linked to recent cis-regulatory changes, particularly in genes involved in lipid metabolism, likely contributing to human-specific metabolic traits. Our study uncovers the origins of a mammal-specific liver cell architecture, within which reduced constraints facilitated molecular changes underlying ecological adaptations.

The retina, whose basic cellular structure is highly conserved across vertebrates, constitutes an accessible system for studying the central nervous system. In recent years, single-cell RNA-sequencing studies have uncovered cellular diversity in the retina of a variety of species, providing new insights on retinal evolution and development. However, similar data in cartilaginous fishes, the sister group to all other extant jawed vertebrates, are still lacking. Here, we present a single-nucleus RNA-sequencing atlas of the postnatal retina of the catshark Scyliorhinus canicula , consisting of the expression profiles for 17,438 individual cells from three female, juvenile catshark specimens. Unsupervised clustering revealed 22 distinct cell types comprising all major retinal cell classes, as well as retinal progenitor cells (whose presence reflects the persistence of proliferative activity in postnatal stages in sharks) and oligodendrocytes. Thus, our dataset serves as a foundation for further studies on the development and function of the catshark retina. Moreover, integration of our atlas with data from other species will allow for a better understanding of vertebrate retinal evolution.

Sexually dimorphic traits are common among mammals and are specified during development through the deployment of sex-specific genetic programs. Because little is known about these programs, we investigated them using a resource of gene expression profiles in males and females throughout the development of five organs in five mammals (human, mouse, rat, rabbit, and opossum) and a bird (chicken). We found that sex-biased gene expression varied considerably across organs and species and was often cell-type specific. Sex differences increased abruptly around sexual maturity instead of increasing gradually during organ development. Finally, sex-biased gene expression evolved rapidly at the gene level, with differences between organs in the evolutionary mechanisms used, but more slowly at the cellular level, with the same cell types being sexually dimorphic across species.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. To understand how GATA4/5/6 modulates the mesoderm-to-cardiac fate transition, we labeled, isolated, and performed single-cell gene expression analysis on cells that expressgata5at precardiac time points spanning zebrafish gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamicgata5/6expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was substantially altered. In addition to the expected absence of cardiac mesoderm, we confirmed a concomitant expansion of cranial-pharyngeal mesoderm. Moreover, Gata5/6 loss led to extensive changes in chromatin accessibility near cardiac and pharyngeal genes. Functional analyses in zebrafish and the tunicateCiona, which has a single GATA4/5/6 homolog, revealed that GATA4/5/6 acts upstream oftbx1to exert essential and cell-autonomous roles in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, cardiac and pharyngeal mesoderm fate choices are achieved through an evolutionarily conserved GATA4/5/6 regulatory network.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. How these factors mediate the transition from multipotent mesoderm progenitors to a committed cardiac fate is unclear. To understand how GATA4/5/6 modulate cell fate decisions we labelled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at pre-cardiac time points spanning gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was dramatically altered. In addition to the expected absence of cardiac mesoderm, we observed a concomitant expansion of cranial-pharyngeal mesoderm. Functional genetic analyses in zebrafish and the invertebrate chordate Ciona , which possess a single GATA4/5/6 homolog, revealed an essential and cell-autonomous role for GATA4/5/6 in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, the maintenance and repression of GATA4/5/6 activity plays a critical, evolutionarily conserved role in early development.

Quebec Platelet Disorder (QPD) is an autosomal dominant bleeding disorder with a unique, platelet-dependent gain-of-function defect in fibrinolysis, without systemic fibrinolysis. The hallmark feature of QPD is a >100-fold overexpression of PLAU specifically in megakaryocytes. This overexpression leads to >100-fold increased platelet stores of urokinase plasminogen activator (PLAU/uPA), subsequent plasmin-mediated degradation of diverse a-granule proteins, and platelet-dependent, accelerated fibrinolysis. The causative mutation is a 78kb tandem duplication of PLAU. How this duplication causes megakaryocyte-specific PLAU overexpression is unknown. To investigate the mechanism that causes QPD, we used epigenomic profiling, comparative genomics, and chromatin conformation capture approaches to study PLAU regulation in cultured megakaryocytes from QPD participants and unaffected controls. We show that the QPD duplication leads to ectopic interactions between PLAU and a conserved megakaryocyte enhancer found within the same topologically associating domain (TAD). Our results support a unique disease mechanism whereby the reorganization of subTAD genome architecture results in a dramatic, cell-type specific blood disorder phenotype.