Emerging roles for diguanylate cyclase during the evolution of soma in dictyostelia.

BACKGROUND: Cyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4. RESULTS: Similar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcA- cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcA- mutants were also observed in acaA- mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcA- cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcA- cells. CONCLUSIONS: The study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcA- is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.

The Origin of Metazoan Multicellularity: A Potential Microbial Black Swan Event.

The emergence of animals from their unicellular ancestors is a major evolutionary event. Thanks to the study of diverse close unicellular relatives of animals, we now have a better grasp of what the unicellular ancestor of animals was like. However, it is unclear how that unicellular ancestor of animals became the first animals. To explain this transition, two popular theories, the choanoblastaea and the synzoospore, have been proposed. We will revise and expose the flaws in these two theories while showing that, due to the limits of our current knowledge, the origin of animals is a biological black swan event. As such, the origin of animals defies retrospective explanations. Therefore, we should be extra careful not to fall for confirmation biases based on few data and, instead, embrace this uncertainty and be open to alternative scenarios. With the aim to broaden the potential explanations on how animals emerged, we here propose two novel and alternative scenarios. In any case, to find the answer to how animals evolved, additional data will be required, as will the hunt for microscopic creatures that are closely related to animals but have not yet been sampled and studied.

Multimerization of HIF enhances transcription of target genes containing the hypoxia ancillary sequence.

Transcriptional activity of the hypoxia inducible factor (HIF) relies on the formation of a heterodimer composed of an oxygen-regulated α-subunit and a stably expressed ß-subunit. Heterodimeric HIF activates expression by binding to RCGTG motifs within promoters of hypoxia-activated genes. Some hypoxia targets also possess an adjacent HIF ancillary sequence (HAS) reported to increase transcription but whose function remains obscure. Here, we investigate the contribution of the HAS element to the hypoxia response and its mechanism of action, using the HAS-containing prolyl 4-hydroxylase subunit α1 (P4HA1) as a gene model in NIH/3T3 mouse embryonic fibroblasts and HEK293 human embryonic kidney cells. Our HIF overexpression experiments demonstrate that the HAS motif is essential for full induction by hypoxia and that the presence of the tandem HAS/HIF, as opposed to HIF-only sequences, provides HIF proteins with the capacity to form complexes of stoichiometry beyond the classical heterodimer, likely tetramers, to cooperatively potentiate hypoxia-induced transcription. We also provide evidence of the crucial role played by the Fα helix of the PAS-B domain of the HIF1ß subunit to support the interaction between heterodimers. Functional analysis showed that human genes containing the HAS/HIF motifs are better responders to hypoxia, and their promoters are enriched for specific transcription factor binding sites. Gene ontology enrichment revealed a predominance of HAS/HIF in genes primarily related to tissue formation and development. Our findings add an extra level of regulation of the hypoxia/HIF signaling through multimerization of HIF proteins on regulatory elements containing the HAS/HIF motifs.

The protein kinases of Dictyostelia and their incorporation into a signalome.

Protein kinases are major regulators of cellular processes, but the roles of most kinases remain unresolved. Dictyostelid social amoebas have been useful in identifying functions for 30% of its kinases in cell migration, cytokinesis, vesicle trafficking, gene regulation and other processes but their upstream regulators and downstream effectors are mostly unknown. Comparative genomics can assist to distinguish between genes involved in deeply conserved core processes and those involved in species-specific innovations, while co-expression of genes as evident from comparative transcriptomics can provide cues to the protein complement of regulatory networks. Genomes and developmental and cell-type specific transcriptomes are available for species that span the 0.5 billion years of evolution of Dictyostelia from their unicellular ancestors. In this work we analysed conservation and change in the abundance, functional domain architecture and developmental regulation of protein kinases across the 4 major taxon groups of Dictyostelia. All data are summarized in annotated phylogenetic trees of the kinase subtypes and accompanied by functional information of all kinases that were experimentally studied. We detected 393 different protein kinase domains across the five studied genomes, of which 212 were fully conserved. Conservation was highest (71%) in the previously defined AGC, CAMK, CK1, CMCG, STE and TKL groups and lowest (26%) in the "other" group of typical protein kinases. This was mostly due to species-specific single gene amplification of "other" kinases. Apart from the AFK and α-kinases, the atypical protein kinases, such as the PIKK and histidine kinases were also almost fully conserved. The phylogeny-wide developmental and cell-type specific expression profiles of the protein kinase genes were combined with profiles from the same transcriptomic experiments for the families of G-protein coupled receptors, small GTPases and their GEFs and GAPs, the transcription factors and for all genes that upon lesion generate a developmental defect. This dataset was subjected to hierarchical clustering to identify clusters of co-expressed genes that potentially act together in a signalling network. The work provides a valuable resource that allows researchers to identify protein kinases and other regulatory proteins that are likely to act as intermediates in a network of interest.

Novel RNAseq-Informed Cell-type Markers and Their Regulation Alter Paradigms of Dictyostelium Developmental Control.

Cell differentiation is traditionally monitored with a few marker genes, which may bias results. To understand the evolution and regulation of the spore, stalk, cup and basal disc cells in Dictyostelia, we previously performed RNAseq on purified cell-types of taxon-group representative dictyostelids. Using promoter-lacZ constructs in D. discoideum, we here investigate the spatio-temporal expression pattern of 29 cell-type specific genes. Genes selected for spore- or cup-specificity in RNAseq were validated as such by lacZ expression, but genes selected for stalk-specificity showed variable additional expression in basal disc, early cup or prestalk populations. We measured responses of 25 genes to 15 single or combined regimes of induction by stimuli known to regulate cell differentiation. The outcomes of these experiments were subjected to hierarchical clustering to identify whether common modes of regulation were correlated with specific expression patterns. The analysis identified a cluster combining the spore and cup genes, which shared upregulation by 8-bromo cyclic AMP and down-regulation by Differentiation Inducing Factor 1 (DIF-1). Most stalk-expressed genes combined into a single cluster and shared strong upregulation by cyclic di-guanylate (c-di-GMP), and synergistic upregulation by combined DIF-1 and c-di-GMP. There was no clustering of genes expressed in other soma besides the stalk, but two genes that were only expressed in the stalk did not respond to any stimuli. In contrast to current models, the study indicates the existence of a stem-cell like soma population in slugs, whose members only acquire ultimate cell fate after progressing to their terminal location during fruiting body morphogenesis.

Evolution of a novel cell type in Dictyostelia required gene duplication of a cudA-like transcription factor.

The evolution of novel cell types has been proposed to result from duplication of gene regulatory networks, but proven examples are rare. In addition to stalk cells and spores that make up the fruiting bodies of three major groups of Dictyostelia, those in group 4 additionally evolved basal disc and cup cells that respectively anchor the stalk to the substratum and the spore mass to the stalk. We noted a putative group-4-specific duplication of a cudA-like transcription factor (TF) in a comparative analysis of group-representative genomes. Using increased taxon sampling, we here confirmed that this TF, cdl1, duplicated into cdl1a and cdl1b in the common ancestor to group 4. cdl1a, but not cdl1b, showed signatures of positive selection, indicative of functional innovation. Deletion of cdl1a in Dictyostelium discoideum resulted in fruiting bodies with sagging spore heads that lacked the supporting cup cells and expression of cup-specific genes. Deletion of cdl1b resulted in thinner fruiting body stalks, while a cdl1b-cdl1a- double knockout showed more severe stalk defects, suggesting an ancestral role of cdl1 in stalk formation. This was confirmed in a closely related non-group 4 species, Polysphondylium violaceum, where cdl1 knockout caused defective stalk formation. These data indicate that the group-specific duplication of cdl1 and subsequent diversification of cdl1a played a pivotal role in the evolution of a novel somatic cell type in group 4 Dictyostelia.

Interactome and evolutionary conservation of Dictyostelid small GTPases and their direct regulators.

GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is under, respectively, positive and negative regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which together with their upstream regulators and the downstream targets of the small GTPases form formidable signalling networks. While genomics has revealed the large size of the GTPase, GEF and GAP repertoires, only a small fraction of their interactions and functions have yet been experimentally explored. Dictyostelid social amoebas have been particularly useful in unravelling the roles of many proteins in the Rac-Rho and Ras-Rap families of GTPases in directional cell migration and regulation of the actin cytoskeleton. Genomes and cell-type specific and developmental transcriptomes are available for Dictyostelium species that span the 0.5 billion years of evolution of the group from their unicellular ancestors. In this work, we identified all GTPases, GEFs and GAPs from genomes representative of the four major taxon groups and investigated their phylogenetic relationships and evolutionary conservation and changes in their functional domain architecture and in their developmental and cell-type specific expression. We performed a hierarchical cluster analysis of the expression profiles of the ~2000 analysed genes to identify putative interacting sets of GTPases, GEFs and GAPs, which highlight sets known to interact experimentally and many novel combinations. This work represents a valuable resource for research into all fields of cellular regulation.

Evolution of Multicellular Complexity in The Dictyostelid Social Amoebas.

Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms, including Dictyostelids, volvocine algae, and metazoans, suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as "proto-cell types" for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the "proto-cell type" gene regulatory networks.

Cold climate adaptation is a plausible cause for evolution of multicellular sporulation in Dictyostelia.

Unicellular protozoa that encyst individually upon starvation evolved at least eight times into organisms that instead form multicellular fruiting bodies with spores. The Dictyostelia are the largest and most complex group of such organisms. They can be subdivided into 4 major groups, with many species in groups 1-3 having additionally retained encystment. To understand fitness differences between spores and cysts, we measured long-term survival of spores and cysts under climate-mimicking conditions, investigated spore and cyst ultrastructure, and related fitness characteristics to species ecology. We found that spores and cysts survived 22 °C equally well, but that spores survived wet and dry frost better than cysts, with group 4 spores being most resilient. Spore walls consist of three layers and those of cysts of maximally two, while spores were also more compacted than cysts, with group 4 spores being the most compacted. Group 4 species were frequently isolated from arctic and alpine zones, which was rarely the case for group 1-3 species. We inferred a fossil-calibrated phylogeny of Dictyostelia, which showed that its two major branches diverged 0.52 billion years ago, following several global glaciations. Our results suggest that Dictyostelium multicellular sporulation was a likely adaptation to a cold climate.

Loss of the Polyketide Synthase StlB Results in Stalk Cell Overproduction in Polysphondylium violaceum.

Major phenotypic innovations in social amoeba evolution occurred at the transition between the Polysphondylia and group 4 Dictyostelia, which comprise the model organism Dictyostelium discoideum, such as the formation of a new structure, the basal disk. Basal disk differentiation and robust stalk formation require the morphogen DIF-1, synthesized by the polyketide synthase StlB, the des-methyl-DIF-1 methyltransferase DmtA, and the chlorinase ChlA, which are conserved throughout Dictyostelia. To understand how the basal disk and other innovations evolved in group 4, we sequenced and annotated the Polysphondylium violaceum (Pvio) genome, performed cell type-specific transcriptomics to identify cell-type marker genes, and developed transformation and gene knock-out procedures for Pvio. We used the novel methods to delete the Pvio stlB gene. The Pvio stlB- mutants formed misshapen curly sorogens with thick and irregular stalks. As fruiting body formation continued, the upper stalks became more regular, but structures contained 40% less spores. The stlB- sorogens overexpressed a stalk gene and underexpressed a (pre)spore gene. Normal fruiting body formation and sporulation were restored in Pvio stlB- by including DIF-1 in the supporting agar. These data indicate that, although conserved, stlB and its product(s) acquired both a novel role in the group 4 Dictyostelia and a role opposite to that in its sister group.

Phylogeny-wide conservation and change in developmental expression, cell-type specificity and functional domains of the transcriptional regulators of social amoebas.

BACKGROUND: Dictyostelid social amoebas self-organize into fruiting bodies, consisting of spores and up to four supporting cell types in the phenotypically most complex taxon group 4. High quality genomes and stage- and cell-type specific transcriptomes are available for representative species of each of the four taxon groups. To understand how evolution of gene regulation in Dictyostelia contributed to evolution of phenotypic complexity, we analysed conservation and change in abundance, functional domain architecture and developmental regulation of their transcription factors (TFs). RESULTS: We detected 440 sequence-specific TFs across 33 families, of which 68% were upregulated in multicellular development and about half conserved throughout Dictyostelia. Prespore cells expressed two times more TFs than prestalk cells, but stalk cells expressed more TFs than spores, suggesting that gene expression events that define spores occur earlier than those that define stalk cells. Changes in TF developmental expression, but not in TF abundance or functional domains occurred more frequently between group 4 and groups 1-3, than between the more distant branches formed by groups 1 + 2 and 3 + 4. CONCLUSIONS: Phenotypic innovation is correlated with changes in TF regulation, rather than functional domain- or TF acquisition. The function of only 34 TFs is known. Of 12 TFs essential for cell differentiation, 9 are expressed in the cell type for which they are required. The information acquired here on conserved cell type specifity of 120 additional TFs can effectively guide further functional analysis, while observed evolutionary change in TF developmental expression may highlight how genotypic change caused phenotypic innovation.

A well supported multi gene phylogeny of 52 dictyostelia.

The Dictyostelid social amoebas are a popular model system for cell- and developmental biology and for evolution of sociality. Small subunit (SSU) ribosomal DNA-based phylogenies subdivide the known 150 species into four major and some minor groups, but lack resolution within groups, particularly group 4, and, as shown by genome-based phylogenies of 11 species, showed errors in the position of the root and nodes separating major clades. We are interested in the evolution of cell-type specialization, which particularly expanded in group 4. To construct a more robust phylogeny, we first included 7 recently sequenced genomes in the genome-based phylogeny of 47 functionally divergent proteins and next selected 6 proteins (Agl, AmdA, PurD, PurL, RpaA, SmdA) that independently or in sets of two fully reproduced the core-phylogeny. We amplified their coding regions from 34 Dictyostelium species and combined their concatenated sequences with those identified in the 18 genomes to generate a fully resolved phylogeny. The new AAPPRS based phylogeny (after the acronym of the 6 proteins) subdivides group 4 into 2 branches. These branches further resolve into 5 clades, rather than the progressively nested group 4 topology of the SSU rDNA tree, and also re-orders taxa in the other major groups. Ancestral state reconstruction of 25 phenotypic traits returned higher "goodness of fit" metrics for evolution of 19 of those traits over the AAPPRS tree, than over the SSU rDNA tree. The novel tree provides a solid framework for studying the evolution of cell-type specialization, signalling and other cellular processes in particularly group 4, which contains the model Dictyostelid D. discoideum.

Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells.

BACKGROUND: A major hallmark of multicellular evolution is increasing complexity by the evolution of new specialized cell types. During Dictyostelid evolution novel specialization occurred within taxon group 4. We here aim to retrace the nature and ancestry of the novel "cup" cells by comparing their transcriptome to that of other cell types. RESULTS: RNA-Seq was performed on purified mature spore, stalk and cup cells and on vegetative amoebas. Clustering and phylogenetic analyses showed that cup cells were most similar to stalk cells, suggesting that they share a common ancestor. The affinity between cup and stalk cells was also evident from promoter-reporter studies of newly identified cell-type genes, which revealed late expression in cups of many stalk genes. However, GO enrichment analysis reveal the unexpected prominence of GTPase mediated signalling in cup cells, in contrast to enrichment of autophagy and cell wall synthesis related transcripts in stalk cells. Combining the cell type RNA-Seq data with developmental expression profiles revealed complex expression dynamics in each cell type as well as genes exclusively expressed during terminal differentiation. Most notable were nine related hssA-like genes that were highly and exclusively expressed in cup cells. CONCLUSIONS: This study reveals the unique transcriptomes of the mature cup, stalk and spore cells of D. discoideum and provides insight into the ancestry of cup cells and roles in signalling that were not previously realized. The data presented in this study will serve as an important resource for future studies into the regulation and evolution of cell type specialization.

The Transcriptomic Evolution of Mammalian Pregnancy: Gene Expression Innovations in Endometrial Stromal Fibroblasts.

The endometrial stromal fibroblast (ESF) is a cell type present in the uterine lining of therian mammals. In the stem lineage of eutherian mammals, ESF acquired the ability to differentiate into decidual cells in order to allow embryo implantation. We call the latter cell type "neo-ESF" in contrast to "paleo-ESF" which is homologous to eutherian ESF but is not able to decidualize. In this study, we compare the transcriptomes of ESF from six therian species: Opossum (Monodelphis domestica; paleo-ESF), mink, rat, rabbit, human (all neo-ESF), and cow (secondarily nondecidualizing neo-ESF). We find evidence for strong stabilizing selection on transcriptome composition suggesting that the expression of approximately 5,600 genes is maintained by natural selection. The evolution of neo-ESF from paleo-ESF involved the following gene expression changes: Loss of expression of genes related to inflammation and immune response, lower expression of genes opposing tissue invasion, increased markers for proliferation as well as the recruitment of FOXM1, a key gene transiently expressed during decidualization. Signaling pathways also evolve rapidly and continue to evolve within eutherian lineages. In the bovine lineage, where invasiveness and decidualization were secondarily lost, we see a re-expression of genes found in opossum, most prominently WISP2, and a loss of gene expression related to angiogenesis. The data from this and previous studies support a scenario, where the proinflammatory paleo-ESF was reprogrammed to express anti-inflammatory genes in response to the inflammatory stimulus coming from the implanting conceptus and thus paving the way for extended, trans-cyclic gestation.

Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles.

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes.

Inferring cell type innovations by phylogenetic methods-concepts, methods, and limitations.

Multicellular organisms are composed of distinct cell types that have specific roles in the body. Each cell type is a product of two kinds of historical processes-development and evolution. Although the concept of a cell type is difficult to define, the cell type concept based on the idea of the core regulatory network (CRN), a gene regulatory network that determines the identity of a cell type, illustrates the essential aspects of the cell type concept. The first step toward elucidating cell type evolution is to reconstruct the evolutionary relationships of cell types, or the cell type tree. The sister cell type model assumes that a new cell type evolves through divergence from a multifunctional ancestral cell type, creating tree-like evolutionary relationships between cell types. The process of generating a cell type tree can also be understood as the sequential addition of a new branching point on an ancestral cell differentiation hierarchy in evolution. A cell type tree thus represents an intertwined history of cell type evolution and development. Cell type trees can be reconstructed from high-throughput sequencing data, and the reconstruction of a cell type tree leads to the discovery of genes that are functionally important for a cell type. Although many issues including the lack of cross-species comparisons and the lack of a proper model for cell type evolution remain, the study of the origin of a new cell type using phylogenetic methods offers a promising new research avenue in developmental evolution. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 653-661, 2015. © 2015 Wiley Periodicals, Inc.

Cell-type phylogenetics and the origin of endometrial stromal cells.

A challenge of genome annotation is the identification of genes performing specific biological functions. Here, we propose a phylogenetic approach that utilizes RNA-seq data to infer the historical relationships among cell types and to trace the pattern of gene-expression changes on the tree. The hypothesis is that gene-expression changes coincidental with the origin of a cell type will be important for the function of the derived cell type. We apply this approach to the endometrial stromal cells (ESCs), which are critical for the initiation and maintenance of pregnancy. Our approach identified well-known regulators of ESCs, PGR and FOXO1, as well as genes not yet implicated in female fertility, including GATA2 and TFAP2C. Knockdown analysis confirmed that they are essential for ESC differentiation. We conclude that phylogenetic analysis of cell transcriptomes is a powerful tool for discovery of genes performing cell-type-specific functions.

Evolution of mammalian pregnancy and the origin of the decidual stromal cell.

Reproduction in eutherian mammals is characterized by extended intrauterine retention of the fetus after implantation. We summarize evolutionary innovations that enable this form of vivipary, including early maternal recognition of pregnancy, invasive placentation, and emergence of the decidual cell type. We first review the structure of the marsupial endometrium and its relationship to that of eutherian mammals. While the tissue components of endometrium are the same in marsupials and eutherians, an important difference is the amount of stromal cells, which are much more abundant in eutherians. Moreover, the nature of the invasive placentation differs in marsupials and eutherians. In the opossum, it consists of cytoplasmatic extensions of trophoblast cells that penetrate between the luminal epithelial cells to contact maternal capillaries. In bandicoots, the trophoblast and luminal epithelial cells fuse, and the maternal epithelium is replaced by a layer of multinucleated cells. In no case has there been evidence of a direct interaction between trophoblast and stromal cells. The direct interface between the trophoblast and maternal stroma is a derived feature of eutherian mammals, coincidental with the origin of decidual cells. Gene expression studies are suggestive of "categorical reprograming" of endometrial fibroblasts during decidualization. This reprogramming suggests that the decidual cell is a distinct cell type rather than a modulation of endometrial fibroblasts. Further support for this hypothesis is the origin of derived transcription factor interactions that are necessary for the regulation of decidual gene expression, in particular the interactions between HOXA11 and CEBPB with FOXO1A.

Immunohistological study of the endometrial stromal fibroblasts in the opossum, Monodelphis domestica: evidence for homology with eutherian stromal fibroblasts.

Molecular phylogenetic studies suggest that the hemochorial placentation and decidualization are ancestral traits of eutherian mammals. While the origin of the placental tissue is well understood, the origin of the decidual cells is unclear. Here we address the origin of decidual cells by examining the expression patterns of six transcription factors (TFs) as well as four structural proteins in the endometrium of a marsupial, Monodelphis domestica, and compared them with the patterns known from eutherian species. We found a mesenchymal cell population in the subepithelial compartment of the opossum endometrium. These cells express a set of TFs, such as homeobox A11 (HOXA11), CCAAT/enhancer-binding protein beta (CEBPB), and progesterone receptor (PGR), that are important for eutherian endometrial stromal cells. On the other hand, we did not find the expression of a decidual cell marker desmin (DES) or of TFs that are important for decidual cell differentiation, such as forkhead box O1 (FOXO1), in those cells. Based on these results, we propose that opossum has cells homologous to eutherian endometrial fibroblasts but no decidual cells. In addition, we describe cellular changes associated with the progression of pregnancy: nuclear localization of CEBPB in luminal epithelial cells as early as 8 days postcoitum, expansion of endometrial glands, nuclear localization of FOXO1 in glandular epithelial cells, and expression of smooth muscle actin in luminal epithelial cells. These data show that the luminal and glandular epithelium react to the presence of the preplacentation conceptus and suggest a limited form of pregnancy recognition.

A model based criterion for gene expression calls using RNA-seq data.

The power of deep sequencing technology to reliably detect single RNA reads leads to a paradoxical problem of high sensitivity. In hybridization or PCR based methods for RNA quantification, the concern is low sensitivity, i.e., the problem that the signal from truly expressed genes might not be distinguishable from noise. In contrast, the problem with RNA-seq is that it is not clear whether genes with very low read counts are from low expressed genes or merely transcriptional noise. The frequency distribution for read counts does not show a clear separation in two classes of genes, which makes the decision whether a gene is to be considered expressed or not seemingly arbitrary. Here we address this problem by suggesting a statistical model that considers the number of transcripts detected in a RNA-seq study as a mixture of two distributions: one is a exponential distribution for transcripts from inactive genes, and a negative binomial distribution for actively transcribed genes. We apply this model to a number of RNA-seq data sets and find that the model fits the data very well. The calculated criteria for distinguishing between expressed and non-expressed gene is remarkably consistent among data sets, suggesting genes with more than two transcripts per million transcripts (TPM) are highly likely from actively transcribed genes. This criterion is consistent with the criterion of 1 RPKM proposed by Hebenstreit et al. Mol Sys Biol 7:497 (2011), based on chromatin modification and per cell RNA expression data. Hence, the regression model correctly identifies the not actively expressed class of genes and thus, provides an operational criterion for classifying genes in expressed and non-expressed sets, facilitating the interpretation of RNA-seq data.