Reframing research on evolutionary novelty and co-option: Character identity mechanisms versus deep homology.

A central topic in research at the intersection of development and evolution is the origin of novel traits. Despite progress on understanding how developmental mechanisms underlie patterns of diversity in the history of life, the problem of novelty continues to challenge researchers. Here we argue that research on evolutionary novelty and the closely associated phenomenon of co-option can be reframed fruitfully by: (1) specifying a conceptual model of mechanisms that underwrite character identity, (2) providing a richer and more empirically precise notion of co-option that goes beyond common appeals to "deep homology", and (3) attending to the nature of experimental interventions that can determine whether and how the co-option of identity mechanisms can help to explain novel character origins. This reframing has the potential to channel future investigation to make substantive progress on the problem of evolutionary novelty. To illustrate this potential, we apply our reframing to two case studies: treehopper helmets and beetle horns.

Cooption of polyalanine tract into a repressor domain in the mammalian transcription factor HoxA11.

An enduring problem in biology is explaining how novel functions of genes originated and how those functions diverge between species. Despite detailed studies on the functional evolution of a few proteins, the molecular mechanisms by which protein functions have evolved are almost entirely unknown. Here, we show that a polyalanine tract in the homeodomain transcription factor HoxA11 arose in the stem-lineage of mammals and functions as an autonomous repressor module by physically interacting with the PAH domains of SIN3 proteins. These results suggest that long polyalanine tracts, which are common in transcription factors and often associated with disease, may tend to function as repressor domains and can contribute to the diversification of transcription factor functions despite the deleterious consequences of polyalanine tract expansion.

Evolution of Embryo Implantation Was Enabled by the Origin of Decidual Stromal Cells in Eutherian Mammals.

Mammalian pregnancy evolved in the therian stem lineage, that is, before the common ancestor of marsupials and eutherian (placental) mammals. Ancestral therian pregnancy likely involved a brief phase of attachment between the fetal and maternal tissues followed by parturition-similar to the situation in most marsupials including the opossum. In all eutherians, however, embryo attachment is followed by implantation, allowing for a stable fetal-maternal interface and an extended gestation. Embryo attachment induces an attachment reaction in the uterus that is homologous to an inflammatory response. Here, we elucidate the evolutionary mechanism by which the ancestral inflammatory response was transformed into embryo implantation in the eutherian lineage. We performed a comparative uterine transcriptomic and immunohistochemical study of three eutherians, armadillo (Dasypus novemcinctus), hyrax (Procavia capensis), and rabbit (Oryctolagus cuniculus); and one marsupial, opossum (Monodelphis domestica). Our results suggest that in the eutherian lineage, the ancestral inflammatory response was domesticated by suppressing one of its modules detrimental to pregnancy, namely, neutrophil recruitment by cytokine IL17A. Further, we propose that this suppression was mediated by decidual stromal cells, a novel cell type in eutherian mammals. We tested a prediction of this model in vitro and showed that decidual stromal cells can suppress the production of IL17A from helper T cells. Together, these results provide a mechanistic understanding of early stages in the evolution of eutherian pregnancy.

Single-cell analysis of prostaglandin E2-induced human decidual cell in vitro differentiation: a minimal ancestral deciduogenic signal†.

The decidua is a hallmark of reproduction in many placental mammals. Differentiation of decidual stromal cells is known to be induced by progesterone and the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Several candidates have been identified as the physiological stimulus for adenylyl cyclase activation, but their relative importance remains unclear. To bypass this uncertainty, the standard approach for in vitro experiments uses membrane-permeable cAMP and progestin. We phylogenetically infer that prostaglandin E2 (PGE2) likely was the signal that ancestrally induced decidualization in conjunction with progesterone. This suggests that PGE2 and progestin should be able to activate the core gene regulatory network of decidual cells. To test this prediction, we performed a genome-wide study of gene expression in human endometrial fibroblasts decidualized with PGE2 and progestin. Comparison to a cAMP-based protocol revealed shared activation of core decidual genes and decreased induction of senescence-associated genes. Single-cell transcriptomics of PGE2-mediated decidualization revealed a distinct, early-activated state transitioning to a differentiated decidual state. PGE2-mediated decidualization was found to depend upon progestin-dependent induction of PGE2 receptor 2 (PTGER2) which in turn leads to PKA activation upon PGE2 stimulation. Progesterone-dependent induction of PTGER2 is absent in opossum, an outgroup taxon of placental mammals which is incapable of decidualization. Together, these findings suggest that the origin of decidualization involved the evolution of progesterone-dependent activation of the PGE2/PTGER2/PKA axis, facilitating entry into a PKA-dominant rather than AKT-dominant cellular state. We propose the use of PGE2 for in vitro decidualization as an alternative to 8-Br-cAMP.

The origin and evolution of cell types.

Cell types are the basic building blocks of multicellular organisms and are extensively diversified in animals. Despite recent advances in characterizing cell types, classification schemes remain ambiguous. We propose an evolutionary definition of a cell type that allows cell types to be delineated and compared within and between species. Key to cell type identity are evolutionary changes in the 'core regulatory complex' (CoRC) of transcription factors, that make emergent sister cell types distinct, enable their independent evolution and regulate cell type-specific traits termed apomeres. We discuss the distinction between developmental and evolutionary lineages, and present a roadmap for future research.

An experimental test of the ovulatory homolog model of female orgasm.

The ovulatory homolog model of female orgasm posits that the neuro-endocrine mechanisms underlying female orgasm evolved from and are homologous to the mechanisms mediating copulation-induced ovulation in some mammals. This model predicts that pharmacological agents that affect human orgasm, such as fluoxetine, should also affect ovulation in animals with copulation-induced ovulation, such as rabbits. We tested this prediction by treating rabbits with daily doses of fluoxetine for 2 wk and found that fluoxetine treatment reduces the number of ovulations postcopulation by 30%. In a second experiment we tested whether this result was mediated by an effect on the brain or via peripheral serotonin functions. We treated animals with fluoxetine and induced ovulation with a single injection of human chorionic gonadotropin. In this experiment ovulation rate was nominally reduced by only 8%, which is statistically not significant. We conclude that the effect of fluoxetine on copulation-induced ovulation rate supports the ovulatory homolog model of female orgasm, suggesting that female orgasm has very deep evolutionary roots among the early eutherian mammals.

Hidden limbs in the "limbless skink" Brachymeles lukbani: Developmental observations.

Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb-reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in Chalcides and limb reduction in Scelotes). The genus Brachymeles, a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake-like species Brachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos of B. lukbani in early stages of development, on the other hand, show a truncated but well-developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole-mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb of B. lukabni. Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well-developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well-developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well-developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that "limbless" lizards that possess these muscles may have or have had at least transient developing limbs, as we find in B. lukbani. Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation called cryptomelia. The results of this work add to our growing understanding of clade-specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes.

The mammalian decidual cell evolved from a cellular stress response.

Among animal species, cell types vary greatly in terms of number and kind. The number of cell types found within an organism differs considerably between species, and cell type diversity is a significant contributor to differences in organismal structure and function. These observations suggest that cell type origination is a significant source of evolutionary novelty. The molecular mechanisms that result in the evolution of novel cell types, however, are poorly understood. Here, we show that a novel cell type of eutherians mammals, the decidual stromal cell (DSC), evolved by rewiring an ancestral cellular stress response. We isolated the precursor cell type of DSCs, endometrial stromal fibroblasts (ESFs), from the opossum Monodelphis domestica. We show that, in opossum ESFs, the majority of decidual core regulatory genes respond to decidualizing signals but do not regulate decidual effector genes. Rather, in opossum ESFs, decidual transcription factors function in apoptotic and oxidative stress response. We propose that rewiring of cellular stress responses was an important mechanism for the evolution of the eutherian decidual cell type.

Stress-Induced Evolutionary Innovation: A Mechanism for the Origin of Cell Types.

Understanding the evolutionary role of environmentally induced phenotypic variation (i.e., plasticity) is an important issue in developmental evolution. A major physiological response to environmental change is cellular stress, which is counteracted by generic stress reactions detoxifying the cell. A model, stress-induced evolutionary innovation (SIEI), whereby ancestral stress reactions and their corresponding pathways can be transformed into novel structural components of body plans, such as new cell types, is described. Previous findings suggest that the cell differentiation cascade of a cell type critical to pregnancy in humans, the decidual stromal cell, evolved from a cellular stress reaction. It is hypothesized that the stress reaction in these cells was elicited ancestrally via inflammation caused by embryo attachment. The present study proposes that SIEI is a distinct form of plasticity-based evolutionary change leading to the origin of novel structures rather than adaptive transformation of pre-existing characters.

Genetic Associations with Gestational Duration and Spontaneous Preterm Birth.

BACKGROUND: Despite evidence that genetic factors contribute to the duration of gestation and the risk of preterm birth, robust associations with genetic variants have not been identified. We used large data sets that included the gestational duration to determine possible genetic associations. METHODS: We performed a genomewide association study in a discovery set of samples obtained from 43,568 women of European ancestry using gestational duration as a continuous trait and term or preterm (<37 weeks) birth as a dichotomous outcome. We used samples from three Nordic data sets (involving a total of 8643 women) to test for replication of genomic loci that had significant genomewide association (P<5.0×10-8) or an association with suggestive significance (P<1.0×10-6) in the discovery set. RESULTS: In the discovery and replication data sets, four loci (EBF1, EEFSEC, AGTR2, and WNT4) were significantly associated with gestational duration. Functional analysis showed that an implicated variant in WNT4 alters the binding of the estrogen receptor. The association between variants in ADCY5 and RAP2C and gestational duration had suggestive significance in the discovery set and significant evidence of association in the replication sets; these variants also showed genomewide significance in a joint analysis. Common variants in EBF1, EEFSEC, and AGTR2 showed association with preterm birth with genomewide significance. An analysis of mother-infant dyads suggested that these variants act at the level of the maternal genome. CONCLUSIONS: In this genomewide association study, we found that variants at the EBF1, EEFSEC, AGTR2, WNT4, ADCY5, and RAP2C loci were associated with gestational duration and variants at the EBF1, EEFSEC, and AGTR2 loci with preterm birth. Previously established roles of these genes in uterine development, maternal nutrition, and vascular control support their mechanistic involvement. (Funded by the March of Dimes and others.).

Single-cell transcriptomics of the human placenta: inferring the cell communication network of the maternal-fetal interface.

Organismal function is, to a great extent, determined by interactions among their fundamental building blocks, the cells. In this work, we studied the cell-cell interactome of fetal placental trophoblast cells and maternal endometrial stromal cells, using single-cell transcriptomics. The placental interface mediates the interaction between two semiallogenic individuals, the mother and the fetus, and is thus the epitome of cell interactions. To study these, we inferred the cell-cell interactome by assessing the gene expression of receptor-ligand pairs across cell types. We find a highly cell-type-specific expression of G-protein-coupled receptors, implying that ligand-receptor profiles could be a reliable tool for cell type identification. Furthermore, we find that uterine decidual cells represent a cell-cell interaction hub with a large number of potential incoming and outgoing signals. Decidual cells differentiate from their precursors, the endometrial stromal fibroblasts, during uterine preparation for pregnancy. We show that decidualization (even in vitro) enhances the ability to communicate with the fetus, as most of the receptors and ligands up-regulated during decidualization have their counterpart expressed in trophoblast cells. Among the signals transmitted, growth factors and immune signals dominate, and suggest a delicate balance of enhancing and suppressive signals. Finally, this study provides a rich resource of gene expression profiles of term intravillous and extravillous trophoblasts, including the transcriptome of the multinucleated syncytiotrophoblast.

Locomotion and palaeoclimate explain the re-evolution of quadrupedal body form in Brachymeles lizards.

Evolutionary reversals, including re-evolution of lost structures, are commonly found in phylogenetic studies. However, we lack an understanding of how these reversals happen mechanistically. A snake-like body form has evolved many times in vertebrates, and occasionally a quadrupedal form has re-evolved, including in Brachymeles lizards. We use body form and locomotion data for species ranging from snake-like to quadrupedal to address how a quadrupedal form could re-evolve. We show that large, quadrupedal species are faster at burying and surface locomotion than snake-like species, indicating a lack of expected performance trade-off between these modes of locomotion. Species with limbs use them while burying, suggesting that limbs are useful for burying in wet, packed substrates. Palaeoclimatological data suggest that Brachymeles originally evolved a snake-like form under a drier climate probably with looser soil in which it was easier to dig. The quadrupedal clade evolved as the climate became humid, where limbs and large size facilitated fossorial locomotion in packed soils.

Transcriptomic responses to hypoxia in endometrial and decidual stromal cells.

Human reproductive success depends on a properly decidualized uterine endometrium that allows implantation and the formation of the placenta. At the core of the decidualization process are endometrial stromal fibroblasts (ESF) that differentiate to decidual stromal cells (DSC). As variations in oxygen levels are functionally relevant in endometrium both upon menstruation and during placentation, we assessed the transcriptomic responses to hypoxia in ESF and DSC. In both cell types, hypoxia-upregulated genes in classical hypoxia pathways such as glycolysis and the epithelial mesenchymal transition. In DSC, hypoxia restored an ESF-like transcriptional state for a subset of transcription factors that are known targets of the progesterone receptor, suggesting that hypoxia partially interferes with progesterone signaling. In both cell types, hypoxia modified transcription of several inflammatory transcription factors that are known regulators of decidualization, including decreased transcription of STATs and increased transcription of CEBPs. We observed that hypoxia-upregulated genes in ESF and DSC had a significant overlap with genes previously detected to be upregulated in endometriotic stromal cells. Promoter analysis of the genes in this overlap suggested the hypoxia-upregulated Jun/Fos and CEBP transcription factors as potential drivers of endometriosis-associated transcription. Using immunohistochemistry, we observed increased expression of JUND and CEBPD in endometriosis lesions compared to healthy endometria. Overall, the findings suggest that hypoxic stress establishes distinct transcriptional states in ESF and DSC and that hypoxia influences the expression of genes that contribute to the core gene regulation of endometriotic stromal cells.

Menstruation: science and society.

Women's health concerns are generally underrepresented in basic and translational research, but reproductive health in particular has been hampered by a lack of understanding of basic uterine and menstrual physiology. Menstrual health is an integral part of overall health because between menarche and menopause, most women menstruate. Yet for tens of millions of women around the world, menstruation regularly and often catastrophically disrupts their physical, mental, and social well-being. Enhancing our understanding of the underlying phenomena involved in menstruation, abnormal uterine bleeding, and other menstruation-related disorders will move us closer to the goal of personalized care. Furthermore, a deeper mechanistic understanding of menstruation-a fast, scarless healing process in healthy individuals-will likely yield insights into a myriad of other diseases involving regulation of vascular function locally and systemically. We also recognize that many women now delay pregnancy and that there is an increasing desire for fertility and uterine preservation. In September 2018, the Gynecologic Health and Disease Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a 2-day meeting, "Menstruation: Science and Society" with an aim to "identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field." Experts in fields ranging from the evolutionary role of menstruation to basic endometrial biology (including omic analysis of the endometrium, stem cells and tissue engineering of the endometrium, endometrial microbiome, and abnormal uterine bleeding and fibroids) and translational medicine (imaging and sampling modalities, patient-focused analysis of menstrual disorders including abnormal uterine bleeding, smart technologies or applications and mobile health platforms) to societal challenges in health literacy and dissemination frameworks across different economic and cultural landscapes shared current state-of-the-art and future vision, incorporating the patient voice at the launch of the meeting. Here, we provide an enhanced meeting report with extensive up-to-date (as of submission) context, capturing the spectrum from how the basic processes of menstruation commence in response to progesterone withdrawal, through the role of tissue-resident and circulating stem and progenitor cells in monthly regeneration-and current gaps in knowledge on how dysregulation leads to abnormal uterine bleeding and other menstruation-related disorders such as adenomyosis, endometriosis, and fibroids-to the clinical challenges in diagnostics, treatment, and patient and societal education. We conclude with an overview of how the global agenda concerning menstruation, and specifically menstrual health and hygiene, are gaining momentum, ranging from increasing investment in addressing menstruation-related barriers facing girls in schools in low- to middle-income countries to the more recent "menstrual equity" and "period poverty" movements spreading across high-income countries.

Tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks.

Alternative inclusion of exons increases the functional diversity of proteins. Among alternatively spliced exons, tissue-specific exons play a critical role in maintaining tissue identity. This raises the question of how tissue-specific protein-coding exons influence protein function. Here we investigate the structural, functional, interaction, and evolutionary properties of constitutive, tissue-specific, and other alternative exons in human. We find that tissue-specific protein segments often contain disordered regions, are enriched in posttranslational modification sites, and frequently embed conserved binding motifs. Furthermore, genes containing tissue-specific exons tend to occupy central positions in interaction networks and display distinct interaction partners in the respective tissues, and are enriched in signaling, development, and disease genes. Based on these findings, we propose that tissue-specific inclusion of disordered segments that contain binding motifs rewires interaction networks and signaling pathways. In this way, tissue-specific splicing may contribute to functional versatility of proteins and increases the diversity of interaction networks across tissues.

Embryo implantation evolved from an ancestral inflammatory attachment reaction.

The molecular changes that support implantation in eutherian mammals are necessary to establish pregnancy. In marsupials, pregnancy is relatively short, and although a placenta does form, it is present for only a few days before parturition. However, morphological changes in the uterus of marsupials at term mimic those that occur during implantation in humans and mice. We investigated the molecular similarity between term pregnancy in the marsupials and implantation in eutherian mammals using the gray short-tailed opossum (Monodelphis domestica) as a model. Transcriptomic analysis shows that term pregnancy in the opossum is characterized by an inflammatory response consistent with implantation in humans and mice. This immune response is temporally correlated with the loss of the eggshell, and we used immunohistochemistry to report that this reaction occurs at the materno-fetal interface. We demonstrate that key markers of implantation, including Heparin binding EGF-like growth factor and Mucin 1, exhibit expression and localization profiles consistent with the pattern observed during implantation in eutherian mammals. Finally, we show that there are transcriptome-wide similarities between the opossum attachment reaction and implantation in rabbits and humans. Our data suggest that the implantation reaction that occurs in eutherians is derived from an attachment reaction in the ancestral therian mammal which, in the opossum, leads directly to parturition. Finally, we argue that the ability to shift from an inflammatory attachment reaction to a noninflammatory period of pregnancy was a key innovation in eutherian mammals that allowed an extended period of intimate placentation.

Endometrial recognition of pregnancy occurs in the grey short-tailed opossum ( Monodelphis domestica).

In human pregnancy, recognition of an embryo within the uterus is essential to support the fetus through gestation. In most marsupials, such as the opossums, pregnancy is shorter than the oestrous cycle and the steroid hormone profile during pregnancy and oestrous cycle are indistinguishable. For these reasons, it was assumed that recognition of pregnancy, as a trait, evolved in the eutherian (placental) stem lineage and independently in wallabies and kangaroos. To investigate whether uterine recognition of pregnancy occurs in species with pregnancy shorter than the oestrous cycle, we examined reproduction in the short-tailed opossum ( Monodelphis domestica), a marsupial with a plesiomorphic mode of pregnancy. We examined the morphological and gene expression changes in the uterus of females in the non-pregnant oestrous cycle and compared these to pregnancy. We found that the presence of an embryo did not alter some aspects of uterine development but increased glandular activity. Transcriptionally, we saw big differences between the uterus of pregnant and cycling animals. These differences included an upregulation of genes involved in transport, inflammation and metabolic-activity in response to the presence of a fetus. Furthermore, transcriptional differences reflected protein level differences in transporter abundance. Our results suggest that while the uterus exhibits programmed changes after ovulation, its transcriptional landscape during pregnancy responds to the presence of a fetus and upregulates genes that may be essential for fetal support. These results are consistent with endometrial recognition of pregnancy occurring in the opossum. While the effects on maternal physiology appear to differ, recognition of pregnancy has now been observed in eutherian mammals, as well as, Australian and American marsupials.

The origin of platelets enabled the evolution of eutherian placentation.

Invasive placentation with extended pregnancy is a shared derived characteristic unique to eutherian mammals that possess a highly effective system of haemostasis, platelets. These are found in all mammals but no other group of animals. We propose that platelets and megakaryocytes (large polyploid nucleated bone marrow cells that produce platelets) evolved from an ancestral 2 N thrombocyte by polyploidization and that the possession of platelets enabled the evolution of invasive placentation. This could explain why invasive placentation is limited to mammals.

Cis-Regulatory Evolution of Forkhead Box O1 (FOXO1), a Terminal Selector Gene for Decidual Stromal Cell Identity.

Studies in human and mouse have shown that decidual stromal cells (DSC), which develop in the innermost lining of uterus, mediate placentation by regulating maternal immune response against the fetus and the extent of fetal invasion. Investigating when and how DSC evolved is thus a key step to reconstructing the evolutionary history of mammalian pregnancy. We present molecular evidence placing the origin of DSC in the stem lineage of eutherians (extant placental mammals). The transcription factor forkhead box O1 (FOXO1) is a part of the core regulatory transcription factor complex (CoRC) that establishes the cell type identity of DSC. Decidualization, the process through which DSC differentiate from endometrial stromal fibroblasts, requires transcriptional upregulation of FOXO1 Contrary to other examples in mammals where gene recruitment is caused by the origin of an alternative promoter, FOXO1 is transcribed from the same promoter in DSC as in endometrial stromal fibroblasts. Comparing the activities of FOXO1 promoters from human, mouse, manatee (Afrotheria), and opossum (marsupial) revealed that FOXO1 promoter evolved responsiveness to decidualization signals in the stem lineage of eutherians. This eutherian vs. marsupial pattern of promoter activity was not observed in some other cell types expressing FOXO1, suggesting that this cis-regulatory evolution occurred specifically in the context of the origin of DSC. Sequence comparison revealed eutherian-specifically conserved nucleotides that contribute to the eutherian promoter activity. We conclude that the cis-regulatory activity of a terminal selector gene for decidual stromal cell identity evolved in the stem lineage of eutherians supporting a model where decidual cells are a eutherian innovation.

The pleiotropic structure of the genotype-phenotype map: the evolvability of complex organisms.

It was first noticed 100 years ago that mutations tend to affect more than one phenotypic characteristic, a phenomenon that was called 'pleiotropy'. Because pleiotropy was found so frequently, the notion arose that pleiotropy is 'universal'. However, quantitative estimates of pleiotropy have not been available until recently. These estimates show that pleiotropy is highly restricted and are more in line with the notion of variational modularity than with universal pleiotropy. This finding has major implications for the evolvability of complex organisms and the mapping of disease-causing mutations.