A single-cell transcriptome atlas of the adult human retina.

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.

Atlas of regulated target genes of transcription factors (ART-TF) in human ES cells.

BACKGROUND: Transcription factors (TFs) play central roles in maintaining "stemness" of embryonic stem (ES) cells and their differentiation into several hundreds of adult cell types. The regulatory competence of TFs is routinely assessed by detecting target genes to which they bind. However, these data do not indicate which target genes are activated, repressed, or not affected by the change of TF abundance. There is a lack of large-scale studies that compare the genome binding of TFs with the expression change of target genes after manipulation of each TF. RESULTS: In this paper we associated human TFs with their target genes by two criteria: binding to genes, evaluated from published ChIP-seq data (n = 1868); and change of target gene expression shortly after induction of each TF in human ES cells. Lists of direction- and strength-specific regulated target genes are generated for 311 TFs (out of 351 TFs tested) with expected proportion of false positives less than or equal to 0.30, including 63 new TFs not present in four existing databases of target genes. Our lists of direction-specific targets for 152 TFs (80.0%) are larger that in the TRRUST database. In average, 30.9% of genes that respond greater than or equal to twofold to the induction of TFs are regulated targets. Regulated target genes indicate that the majority of TFs are either strong activators or strong repressors, whereas sets of genes that responded greater than or equal to twofold to the induction of TFs did not show strong asymmetry in the direction of expression change. The majority of human TFs (82.1%) regulated their target genes primarily via binding to enhancers. Repression of target genes is more often mediated by promoter-binding than activation of target genes. Enhancer-promoter loops are more abundant among strong activator and repressor TFs. CONCLUSIONS: We developed an atlas of regulated targets of TFs (ART-TF) in human ES cells by combining data on TF binding with data on gene expression change after manipulation of individual TFs. Sets of regulated gene targets were identified with a controlled rate of false positives. This approach contributes to the understanding of biological functions of TFs and organization of gene regulatory networks. This atlas should be a valuable resource for ES cell-based regenerative medicine studies.

Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells.

The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.

Functional heterogeneity of embryonic stem cells revealed through translational amplification of an early endodermal transcript.

ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically "undifferentiated" cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V(+)S(+)), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly when introduced back into morulae or blastocysts, the V(+)S(+) population is not effective at contributing to the epiblast and can contribute to the extra-embryonic visceral and parietal endoderm, while the V(-)S(+) population generates high contribution chimeras. Taken together our data support a model in which ES cell culture has trapped a set of interconvertible cell states reminiscent of the early stages in blastocyst differentiation that may exist only transiently in the early embryo.

Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity.

BACKGROUND: In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before. RESULTS: We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (N=4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (N=9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters. CONCLUSIONS: We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity.

A role for borg5 during trophectoderm differentiation.

Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5'-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development.

Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/-) embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/-) embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk.

Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(-) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(-) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60% increase in premalignant aberrant crypt foci (ACF) formation over LOI(-) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30% lower even than found in exposed LOI(-) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are "IGF-II addicted" and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade.

Functional Information: Towards Synthesis of Biosemiotics and Cybernetics.

Biosemiotics and cybernetics are closely related, yet they are separated by the boundary between life and non-life: biosemiotics is focused on living organisms, whereas cybernetics is applied mostly to non-living artificial devices. However, both classes of systems are agents that perform functions necessary for reaching their goals. I propose to shift the focus of biosemiotics from living organisms to agents in general, which all belong to a pragmasphere or functional universe. Agents should be considered in the context of their hierarchy and origin because their semiosis can be inherited or induced by higher-level agents. To preserve and disseminate their functions, agents use functional information - a set of signs that encode and control their functions. It includes stable memory signs, transient messengers, and natural signs. The origin and evolution of functional information is discussed in terms of transitions between vegetative, animal, and social levels of semiosis, defined by Kull. Vegetative semiosis differs substantially from higher levels of semiosis, because signs are recognized and interpreted via direct code-based matching and are not associated with ideal representations of objects. Thus, I consider a separate classification of signs at the vegetative level that includes proto-icons, proto-indexes, and proto-symbols. Animal and social semiosis are based on classification, and modeling of objects, which represent the knowledge of agents about their body (Innenwelt) and environment (Umwelt).

Generation and Profiling of 2,135 Human ESC Lines for the Systematic Analyses of Cell States Perturbed by Inducing Single Transcription Factors.

Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.

Dynamics of global gene expression changes during mouse preimplantation development.

Understanding preimplantation development is important both for basic reproductive biology and for practical applications including regenerative medicine and livestock breeding. Global expression profiles revealed and characterized the distinctive patterns of maternal RNA degradation and zygotic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA); the second wave, named mid-preimplantation gene activation (MGA), precedes the dynamic morphological and functional changes from the morula to blastocyst stage. Further expression profiling of embryos treated with inhibitors of transcription, translation, and DNA replication revealed that the translation of maternal RNAs is required for the initiation of ZGA. We propose a cascade of gene activation from maternal RNA/protein sets to ZGA gene sets and thence to MGA gene sets. The large number of genes identified as involved in each phase is a first step toward analysis of the complex gene regulatory networks.

BAF250B-associated SWI/SNF chromatin-remodeling complex is required to maintain undifferentiated mouse embryonic stem cells.

Whether SWI/SNF chromatin remodeling complexes play roles in embryonic stem (ES) cells remains unknown. Here we show that SWI/SNF complexes are present in mouse ES cells, and their composition is dynamically regulated upon induction of ES cell differentiation. For example, the SWI/SNF purified from undifferentiated ES cells contains a high level of BAF155 and a low level of BAF170 (both of which are homologs of yeast SWI3 protein), whereas that from differentiated cells contains nearly equal amounts of both. Moreover, the levels of BAF250A and BAF250B decrease during the differentiation of ES cells, whereas that of BRM increases. The altered expression of SWI/SNF components hinted that these complexes could play roles in ES cell maintenance or differentiation. We therefore generated ES cells with biallelic inactivation of BAF250B and found that these cells display a reduced proliferation rate and an abnormal cell cycle. Importantly, these cells are deficient in the self-renewal capacity of undifferentiated ES cells and exhibit certain phenotypes of differentiated cells, including reduced expression of several pluripotency-related genes and increased expression of some differentiation-related genes. These data suggest that the BAF250B-associated SWI/SNF is essential for mouse ES cells to maintain their normal proliferation and pluripotency. The work presented here underscores the importance of SWI/SNF chromatin remodeling complexes in pluripotent stem cells.

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary.

BACKGROUND: The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR). RESULTS: We found that gene expression changes occurred in aging gonads, but were generally different from those in somatic organs during aging. For example, only two functional categories of genes previously associated with aging in muscle, kidney, and brain were confirmed in ovary: genes associated with complement activation were upregulated, and genes associated with mitochondrial electron transport were downregulated. The bulk of the changes in gonads were mostly related to gonad-specific functions. Ovaries showed extensive gene expression changes with age, especially in the period when ovulation ceases (from 6 to 16 months), whereas testes showed only limited age-related changes. The same trend was seen for the effects of CR: CR-mediated reversal of age-associated gene expression changes, reported in somatic organs previously, was limited to a small number of genes in gonads. Instead, in both ovary and testis, CR caused small and mostly gonad-specific effects: suppression of ovulation in ovary and activation of testis-specific genes in testis. CONCLUSION: Overall, the results are consistent with unique modes of aging and its modification by CR in testis and ovary.

Coenzyme autocatalytic network on the surface of oil microspheres as a model for the origin of life.

Coenzymes are often considered as remnants of primordial metabolism, but not as hereditary molecules. I suggest that coenzyme-like molecules (CLMs) performed hereditary functions before the emergence of nucleic acids. Autocatalytic CLMs modified (encoded) surface properties of hydrocarbon microspheres, to which they were anchored, and these changes enhanced autocatalysis and propagation of CLMs. Heredity started from a single kind of self-reproducing CLM, and then evolved into more complex coenzyme autocatalytic networks containing multiple kinds of CLMs. Polymerization of CLMs on the surface of microspheres and development of template-based synthesis is a potential evolutionary path towards the emergence of nucleic acids.

Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data.

BACKGROUND: Target genes of a transcription factor (TF) Pou5f1 (Oct3/4 or Oct4), which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES) cells, have previously been identified based on their response to Pou5f1 manipulation and occurrence of Chromatin-immunoprecipitation (ChIP)-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation. RESULTS: To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR) criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR < 0.2) to a compendium of published and new microarray data (3, 6, 12, and 24 hr after Pou5f1 suppression) and published ChIP data, we identified 420 tentative target genes (TTGs) for Pou5f1. The majority of TTGs (372) were down-regulated after Pou5f1 suppression, indicating that the Pou5f1 functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that Sox2 and Nanog also function mostly as transcription activators in cooperation with Pou5f1. CONCLUSION: We have identified the most reliable sets of direct target genes for key pluripotency genes - Pou5f1, Sox2, and Nanog, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly.

Transcriptome analysis of mouse stem cells and early embryos.

Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

Composite Agency: Semiotics of Modularity and Guiding Interactions.

Principles of constructivism are used here to explore how organisms develop tools, subagents, scaffolds, signs, and adaptations. Here I discuss reasons why organisms have composite nature and include diverse subagents that interact in partially cooperating and partially conflicting ways. Such modularity is necessary for efficient and robust functionality, including mutual construction and adaptability at various time scales. Subagents interact via material and semiotic relations, some of which force or prescribe actions of partners. Other interactions, which I call "guiding", do not have immediate effects and do not disrupt the evolution and learning capacity of partner agents. However, they modify the extent of learning and evolutionary possibilities of partners via establishment of scaffolds and constraints. As a result, subagents construct reciprocal scaffolding for each other to rebalance their communal evolution and learning. As an example, I discuss guiding interactions between the body and mind of animals, where the pain system adjusts mind-based learning to the physical and physiological constraints of the body. Reciprocal effects of mind and behaviors on the development and evolution of the body includes the effects of Lamarck and Baldwin.

CisView: a browser and database of cis-regulatory modules predicted in the mouse genome.

To facilitate the analysis of gene regulatory regions of the mouse genome, we developed a CisView (http://lgsun.grc.nia.nih.gov/cisview), a browser and database of genome-wide potential transcription factor binding sites (TFBSs) that were identified using 134 position-weight matrices and 219 sequence patterns from various sources and were presented with the information about sequence conservation, neighboring genes and their structures, GO annotations, protein domains, DNA repeats and CpG islands. Analysis of the distribution of TFBSs revealed that many TFBSs (N = 145) were over-represented near transcription start sites. We also identified potential cis-regulatory modules (CRMs) defined as clusters of conserved TFBSs in the entire mouse genome. Out of 739 074 CRMs, 157 442 had a significantly higher regulatory potential score than semi-random sequences generated with a 3rd-order Markov process. The CisView browser provides a user-friendly computer environment for studying transcription regulation on a whole-genome scale and can also be used for interpreting microarray experiments and identifying putative targets of transcription factors.

Coenzyme world model of the origin of life.

The origin of life means the emergence of heritable and evolvable self-reproduction. However the mechanisms of primordial heredity were different from those in contemporary cells. Here I argue that primordial life had no nucleic acids; instead heritable signs were represented by isolated catalytically active self-reproducing molecules, similar to extant coenzymes, which presumably colonized surfaces of oil droplets in water. The model further assumes that coenzyme-like molecules (CLMs) changed surface properties of oil droplets (e.g., by oxidizing terminal carbons), and in this way created and sustained favorable conditions for their own self-reproduction. Such niche-dependent self-reproduction is a necessary condition for cooperation between different kinds of CLMs because they have to coexist in the same oil droplets and either succeed or perish together. Additional kinds of hereditary molecules were acquired via coalescence of oil droplets carrying different kinds of CLMs or via modification of already existing CLMs. Eventually, polymerization of CLMs became controlled by other polymers used as templates; and this kind of template-based synthesis eventually resulted in the emergence of RNA-like replicons. Apparently, oil droplets transformed into the outer membrane of cells via engulfing water, stabilization of the surface, and osmoregulation. In result, the metabolism was internalized allowing cells to accumulate free-floating resources (e.g., animoacids, ATP), which was a necessary condition for the development of protein synthesis. Thus, life originated from simple but already functional molecules, and its gradual evolution towards higher complexity was driven by cooperation and natural selection.

Evolutionary biosemiotics and multilevel construction networks.

In contrast to the traditional relational semiotics, biosemiotics decisively deviates towards dynamical aspects of signs at the evolutionary and developmental time scales. The analysis of sign dynamics requires constructivism (in a broad sense) to explain how new components such as subagents, sensors, effectors, and interpretation networks are produced by developing and evolving organisms. Semiotic networks that include signs, tools, and subagents are multilevel, and this feature supports the plasticity, robustness, and evolvability of organisms. The origin of life is described here as the emergence of simple self-constructing semiotic networks that progressively increased the diversity of their components and relations. Primitive organisms have no capacity to classify and track objects; thus, we need to admit the existence of proto-signs that directly regulate activities of agents without being associated with objects. However, object recognition and handling became possible in eukaryotic species with the development of extensive rewritable epigenetic memory as well as sensorial and effector capacities. Semiotic networks are based on sequential and recursive construction, where each step produces components (i.e., agents, scaffolds, signs, and resources) that are needed for the following steps of construction. Construction is not limited to repair and reproduction of what already exists or is unambiguously encoded, it also includes production of new components and behaviors via learning and evolution. A special case is the emergence of new levels of organization known as metasystem transition. Multilevel semiotic networks reshape the phenotype of organisms by combining a mosaic of features developed via learning and evolution of cooperating and/or conflicting subagents.