How fetal and maternal cell types have co-evolved to enable mammalian placentation poses a unique evolutionary puzzle. Here we integrate and compare single-cell transcriptomes from six species bracketing therian mammal diversity: opossum (a marsupial), Malagasy common tenrec (an afrotherian), mouse and guinea pig (rodents), and macaque and human (primates). We identify a conserved transcriptomic signature of invasive trophoblast across eutherians, probably representing a cell type family that radiated with the evolution of haemochorial placentation. In the maternal stroma, comparative analysis reveals that the endocrine decidual cell evolved from an immunomodulatory predecidual cell type retained in Tenrec and resembling early human decidua. Fetal and maternal cell signalling shows a pronounced tendency towards disambiguation—the exclusive expression of ligands by only one partner—although few ligand–receptor pairs follow an escalatory arms race dynamic. Finally, we reconstruct the uteroplacental cell–cell communication networks of extinct mammalian ancestors, identifying signalling innovations and widespread integration of fetal trophoblast and maternal decidual cells into signalling networks. Together, these results reveal a dynamic history of cell type innovation and co-evolution at the fetal–maternal interface.

Evolutionary profiling has been largely limited to the nucleotide level. Using consistent proteomic methods, we quantified proteomic and phosphoproteomic layers in fibroblasts from 11 common mammalian species, with transcriptomes as reference. Covariation analysis indicates that transcript and protein expression levels and variabilities across mammals remarkably follow functional role, with extracellular matrix–associated expression being the most variable, demonstrating strong transcriptome-proteome coevolution. The biological variability of gene expression is universal at both interindividual and interspecies scales but to a different extent. RNA metabolic processes particularly show higher interspecies versus interindividual variation. Our results further indicate that while the ubiquitin-proteasome system is strongly conserved in mammals, lysosome-mediated protein degradation exhibits remarkable variation between mammalian lineages. In addition, the phosphosite profiles reveal a phosphorylation coevolution network independent of protein abundance.

Males have evolved species-specifical sperm morphology and swimming patterns to adapt to different fertilization environments. In eutherians, only a small fraction of the sperm overcome the diverse obstacles in the female reproductive tract and successfully migrate to the fertilizing site. Sperm arriving at the fertilizing site show hyperactivated motility, a unique motility pattern displaying asymmetric beating of sperm flagella with increased amplitude. This motility change is triggered by Ca²⁺ influx through the sperm-specific ion channel, CatSper. However, the current understanding of the CatSper function and its molecular regulation is limited in eutherians. Here, we report molecular evolution and conservation of the CatSper channel in the genome throughout eutherians and marsupials. Sequence analyses reveal that CatSper proteins are slowly evolved in marsupials. Using an American marsupial, gray short-tailed opossum (Monodelphis domestica), we demonstrate the expression of CatSper in testes and its function in hyperactivation and unpairing of sperm. We demonstrate that a conserved IQ-like motif in CatSperζ is required for CatSperζ interaction with the pH-tuned Ca²⁺ sensor, EFCAB9, for regulating CatSper activity. Recombinant opossum EFCAB9 can interact with mouse CatSperζ despite high sequence divergence of CatSperζ among CatSper subunits in therians. Our finding suggests that molecular characteristics and functions of CatSper are evolutionarily conserved in gray short-tailed opossum, unraveling the significance of sperm hyperactivation and fertilization in marsupials for the first time.

Eutherian (placental) mammals exhibit great differences in the degree of placental invasion into the maternal endometrium, with humans being on the most invasive end. Previously, we have shown that these differences in invasiveness is largely controlled by the stromal fibroblasts of the maternal endometrium, with secondary effect on stroma of other tissues resulting in correlated differences in cancer malignancy. Here, we present a statistical investigation of the second dogma linking the phenotypic and transcriptional differences to the genomic changes across species, revealing the regulatory genomic sequence differences underlying these inter-species differences. We show that gain or loss of specific transcription factor binding site sequences are connected to the inter-species gene-expression differences in a statistically significant manner, with a particularly larger effect on stromal genes related to invasibility. We also uncover transcriptional factors differentially regulating genes related to pro- and anti- invasible property of stroma. This work extends the understanding of inter-species differences in stromal invasion to the causal genomic sequence differences paving new avenues to target stromal characteristics to regulate placental, or cancer invasion.

CD44 is a membrane-bound extracellular matrix (ECM) receptor interacting, among others, with hyaluronic acid (HA) and osteopontin (OPN). Cancer progression and metastasis are greatly influenced by the cancer micro-environment, consisting of ECM, immune cells and cancer-associated fibroblasts (CAF). Recruitment of fibroblasts (FB) into the role as CAFs is caused by paracrine signals from the tumor, including TGFb1, PDGF and OPN. The effect of OPN on the transformation of FB into CAF is mediated by CD44. CD44 expression in human skin and endometrial stromal fibroblasts (SF and ESF, respectively) also enhances invasibility of stroma by trophoblast as well as cancer cells. Here we study the evolution of CD44 expression in therian mammals in both SF as well as ESF and demonstrate that the human lineage has experienced a concerted evolutionary enhancement of CD44 expression in SF and ESF, correlating with an increase in human vulnerability to cancer malignancy. In both human and cattle ( Bos taurus ), the dominant isoforms are CD44s and CD44v10 with 9 and 10 exons, respectively. CD44s is an isoform strongly associated with malignancy. In humans, an additional isoform is expressed: HsaCD44-205 with 8 exons not found in cattle. We show that the concerted increase of CD44 expression in SF and ESF is largely due to cis-regulatory effects in the proximal promoter of CD44 . We identify a primate specific acquisition of CEBPB binding sites in the CD44 promoter. Recruitment of CEBPB into CD44 regulation explains almost 50% of the lineage-specific increased CD44 expression in primate skin fibroblasts but is not necessary for high CD44 expression in ESF. All these results suggest that selective modulation of CD44 expression in skin fibroblasts could attenuate the cancer-promoting effect of CAF recruitment in the skin with minimal side effects on other cell types. Additional experimental data is needed to explore this possibility.

Chemical neurotransmission was a key animal innovation, enabling multicellular coordination of physiology and locomotion. Sponges are early-diverging animals that lack neurons and muscles, yet still coordinate contraction and relaxation of their filter-feeding water canals. Here, we show that Spongilla lacustris synthesizes the monoamines tryptamine, phenethylamine, and tyramine to elicit distinct canal behaviors. We identify previously uncharacterized decarboxylases and vesicular transporters coexpressed in secretory neuroid and metabolic cells. Using phosphoproteomics and label-free 3D imaging, we show that tryptamine activates GPCR signaling and Rho GTPases, remodeling adhesion and actomyosin networks in contractile canal epithelia to drive localized constrictions and whole-body deflations. Together, these findings define an ancestral monoaminergic system linking secretory and contractile cell types that predate neurons and was later elaborated for neuromodulation of synaptic transmission.