To be or not to be: The active inference of suicide.

Suicide presents an apparent paradox as a behavior whose motivation is not obvious since its outcome is non-existence and cannot be experienced. To address this paradox, we propose to frame suicide in the integrated theory of stress and active inference. We present an active inference-based cognitive model of suicide as a type of stress response hanging in cognitive balance between predicting self-preservation and self-destruction. In it, self-efficacy emerges as a meta-cognitive regulator that can bias the model toward either survival or suicide. The model suggests conditions under which cognitive homeostasis can override physiological homeostasis in motivating self-destruction. We also present a model proto-suicidal behavior, programmed cell death (apoptosis), in active inference terms to illustrate how an active inference model of self-destruction can be embodied in molecular mechanisms and to offer a hypothesis on another puzzle of suicide: why only humans among brain-endowed animals are known to practice it.

Innate immune system activation in zebrafish and cellular models of Diamond Blackfan Anemia.

Deficiency of ribosomal proteins (RPs) leads to Diamond Blackfan Anemia (DBA) associated with anemia, congenital defects, and cancer. While p53 activation is responsible for many features of DBA, the role of immune system is less defined. The Innate immune system can be activated by endogenous nucleic acids from non-processed pre-rRNAs, DNA damage, and apoptosis that occurs in DBA. Recognition by toll like receptors (TLRs) and Mda5-like sensors induces interferons (IFNs) and inflammation. Dying cells can also activate complement system. Therefore we analyzed the status of these pathways in RP-deficient zebrafish and found upregulation of interferon, inflammatory cytokines and mediators, and complement. We also found upregulation of receptors signaling to IFNs including Mda5, Tlr3, and Tlr9. TGFb family member activin was also upregulated in RP-deficient zebrafish and in RPS19-deficient human cells, which include a lymphoid cell line from a DBA patient, and fetal liver cells and K562 cells transduced with RPS19 shRNA. Treatment of RP-deficient zebrafish with a TLR3 inhibitor decreased IFNs activation, acute phase response, and apoptosis and improved their hematopoiesis and morphology. Inhibitors of complement and activin also had beneficial effects. Our studies suggest that innate immune system contributes to the phenotype of RPS19-deficient zebrafish and human cells.

Ribosomopathies: how a common root can cause a tree of pathologies.

Defects in ribosome biogenesis are associated with a group of diseases called the ribosomopathies, of which Diamond-Blackfan anemia (DBA) is the most studied. Ribosomes are composed of ribosomal proteins (RPs) and ribosomal RNA (rRNA). RPs and multiple other factors are necessary for the processing of pre-rRNA, the assembly of ribosomal subunits, their export to the cytoplasm and for the final assembly of subunits into a ribosome. Haploinsufficiency of certain RPs causes DBA, whereas mutations in other factors cause various other ribosomopathies. Despite the general nature of their underlying defects, the clinical manifestations of ribosomopathies differ. In DBA, for example, red blood cell pathology is especially evident. In addition, individuals with DBA often have malformations of limbs, the face and various organs, and also have an increased risk of cancer. Common features shared among human DBA and animal models have emerged, such as small body size, eye defects, duplication or overgrowth of ectoderm-derived structures, and hematopoietic defects. Phenotypes of ribosomopathies are mediated both by p53-dependent and -independent pathways. The current challenge is to identify differences in response to ribosomal stress that lead to specific tissue defects in various ribosomopathies. Here, we review recent findings in this field, with a particular focus on animal models, and discuss how, in some cases, the different phenotypes of ribosomopathies might arise from differences in the spatiotemporal expression of the affected genes.

TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors.

Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-α (TNF-α) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

The evolution of adaptive immunity.

The concept of adaptive immunity suggests de novo generation in each individual of extremely large repertoires of diversified receptors and selective expansion of receptors that match the antigen/pathogen. Accordingly, adaptive immune system is also called "anticipatory". It allows each individual to have a unique repertoire of immune receptors corresponding to its life history. The memory of an antigen gets encoded in the clonal composition of the organism's immune cells instead of being encoded in the genome. Consequently, the immune response to repeated encounter with the same antigen becomes stronger, a phenomenon called immunological memory. Elements of adaptive immunity are found at all taxonomical levels, whereas in vertebrates, adaptive mechanisms have become the cornerstone of the immune system. In jaw vertebrates, adaptive immune receptors of T and B lymphoid cells belong to immunoglobulin superfamily and are created by rearrangement of gene segments. In jawless vertebrates lamprey and hagfish, recombination of leucine-rich repeat modules is used to form variable lymphocyte receptors. Striking functional similarity of the cellular and humoral branches of these systems suggests similar driving forces underlying their development.

Zebrafish models for dyskeratosis congenita reveal critical roles of p53 activation contributing to hematopoietic defects through RNA processing.

Dyskeratosis congenita (DC) is a rare bone marrow failure syndrome in which hematopoietic defects are the main cause of mortality. The most studied gene responsible for DC pathogenesis is DKC1 while mutations in several other genes encoding components of the H/ACA RNP telomerase complex, which is involved in ribosomal RNA(rRNA) processing and telomere maintenance, have also been implicated. GAR1/nola1 is one of the four core proteins of the H/ACA RNP complex. Through comparative analysis of morpholino oligonucleotide induced knockdown of dkc1 and a retrovirus insertion induced mutation of GAR1/nola1 in zebrafish, we demonstrate that hematopoietic defects are specifically recapitulated in these models and that these defects are significantly reduced in a p53 null mutant background. We further show that changes in telomerase activity are undetectable at the early stages of DC pathogenesis but rRNA processing is clearly defective. Our data therefore support a model that deficiency in dkc1 and nola1 in the H/ACA RNP complex likely contributes to the hematopoietic phenotype through p53 activation associated with rRNA processing defects rather than telomerase deficiency during the initial stage of DC pathogenesis.

Ribosomal protein L11 mutation in zebrafish leads to haematopoietic and metabolic defects.

Mutations in ribosomal proteins are associated with a congenital syndrome, Diamond-Blackfan anaemia (DBA), manifested by red blood cell aplasia, developmental abnormalities and increased risk of malignancy. Recent studies suggest the involvement of p53 activation in DBA. However, which pathways are involved and how they contribute to the DBA phenotype remains unknown. Here we show that a zebrafish mutant for the rpl11 gene had defects both in the development of haematopoietic stem cells (HSCs) and maintenance of erythroid cells. The molecular signature of the mutant included upregulation of p53 target genes and global changes in metabolism. The changes in several pathways may affect haematopoiesis including upregulation of pro-apoptotic and cell cycle arrest genes, suppression of glycolysis, downregulation of biosynthesis and dysregulation of cytoskeleton. Each of these pathways has been individually implicated in haematological diseases. Inhibition of p53 partially rescued haematopoiesis in the mutant. Altogether, we propose that the unique phenotype of DBA is a sum of several abnormally regulated molecular pathways, mediated by the p53 protein family and p53-independent, which have synergistic impact on haematological and other cellular pathways affected in DBA. Our results provide new insights into the pathogenesis of DBA and point to the potential avenues for therapeutic intervention.

p53 upregulation is a frequent response to deficiency of cell-essential genes.

BACKGROUND: The role of p53 in the prevention of development of embryos damaged by genotoxic factors is well recognized. However, whether p53 plays an analogous role in preventing birth defects from genetic mutations remains an unanswered question. Genetic screens for mutations affecting development show that only a fraction of developmentally lethal mutations leads to specific phenotypes while the majority results in similar recurrent phenotypes characterized by neuronal apoptosis and developmental delay. Mutations in cell-essential genes typically fall into this group. The observation that mutations in diverse housekeeping genes lead to a similar phenotype suggests a common mechanism underlying this phenotype. For some mutants, p53 inhibition was shown to attenuate the phenotype. METHODOLOGY/PRINCIPAL FINDINGS: To find out how common p53 involvement is in this phenotype, we analyzed zebrafish mutants from various categories of cell essential genes. Several thousand zebrafish mutants have been identified; many of them are kept at stock centers and available for the research community. We selected mutants for genes functioning in DNA replication, transcription, telomere maintenance, ribosome biogenesis, splicing, chaperoning, endocytosis, and cellular transport. We found that mutants have similar phenotypes including neural apoptosis, failure to develop structures originated from the neural crest cells, and hematopoietic defects. All mutants share p53 upregulation and similar changes in several p53-dependent and independent molecular pathways. CONCLUSION/SIGNIFICANCE: Our results suggest that mutations in housekeeping genes often canalize on the p53-mediated phenotype. p53 prevents the development of embryos with defects in such genes. p53-mediated changes in gene expression may also contribute to many human congenital malformations.

Going adaptive: the saga of antibodies.

Because of their extreme importance to human health, we probably know more about the structure and function of antibodies than practically any other molecule. Despite all the knowledge that has been accrued in the understanding of antibodies, modern approaches, especially comparative genomics, continue to yield novel findings regarding their underlying biology and evolution. In this review, we describe recent research that led to these revelations, and discuss the broad evolutionary implications of these findings. We have restricted our discussion to three vignettes. Considerable attention has been paid to the recent discovery that the teleost IgH locus is highly similar in organization to the Tcra-Tcrd locus, implicating an evolutionary common ancestor and parallels between the functions of B and T cells during development. Second, we discuss how a new type of antibody, recently discovered in jawless vertebrates, composed not of immunoglobulins but leucine-rich repeats, sheds new light on the overall forces driving evolution of all adaptive antigen receptors. Lastly, we discuss how accumulation of genomic sequences of various human subpopulations leads to better understanding of the directionality of antibody evolution. There is always more to learn from the unfolding saga of antibodies.

p53 family in development.

The p53 family network is a unique cellular processor that integrates information from various pathways and determines cellular choices between proliferation, replication arrest/repair, differentiation, senescence, or apoptosis. The most studied role of the p53 family is the regulation of stress response and tumor suppression. By removing damaged cells from the proliferating pool, p53 family members preserve the integrity of the genome. In addition to this well recognized role, recent data implicate the p53 protein family in a broader role of controlling cell proliferation, differentiation and death. Members of the p53 protein family with opposing activity perform coordination of these processes. Imbalance of p53 protein family may contribute to a significant proportion of congenital developmental abnormalities in humans.

Role of p53 family in birth defects: lessons from zebrafish.

p53 Protein family is an important teratologic suppressor, but in certain conditions it can cause congenital abnormalities. p53 Family performs this dual role in development by integrating information from cell's interior with that from the environment to determine the choice between life and death. Understanding of p53 family developmental functions may lead to new therapeutic approaches for treatment and prevention of birth defects. Zebrafish is becoming the vertebrate system of choice for studying p53 family role in development.

Ribosomal protein S19 deficiency in zebrafish leads to developmental abnormalities and defective erythropoiesis through activation of p53 protein family.

Mutations in several ribosomal proteins (RPs) lead to Diamond-Blackfan anemia (DBA), a syndrome characterized by defective erythropoiesis, congenital anomalies, and increased frequency of cancer. RPS19 is the most frequently mutated RP in DBA. RPS19 deficiency impairs ribosomal biogenesis, but how this leads to DBA or cancer remains unknown. We have found that rps19 deficiency in ze-brafish results in hematopoietic and developmental abnormalities resembling DBA. Our data suggest that the rps19-deficient phenotype is mediated by dysregulation of deltaNp63 and p53. During gastrulation, deltaNp63 is required for specification of nonneural ectoderm and its up-regulation suppresses neural differentiation, thus contributing to brain/craniofacial defects. In rps19-deficient embryos, deltaNp63 is induced in erythroid progenitors and may contribute to blood defects. We have shown that suppression of p53 and deltaNp63 alleviates the rps19-deficient phenotypes. Mutations in other ribosomal proteins, such as S8, S11, and S18, also lead to up-regulation of p53 pathway, suggesting it is a common response to ribosomal protein deficiency. Our finding provides new insights into pathogenesis of DBA. Ribosomal stress syndromes represent a broader spectrum of human congenital diseases caused by genotoxic stress; therefore, imbalance of p53 family members may become a new target for therapeutics.

The evolution of immune mechanisms.

From early on in evolution, organisms have had to protect themselves from pathogens. Mechanisms for discriminating "self" from "non-self" evolved to accomplish this task, launching a long history of host-pathogen co-evolution. Evolution of mechanisms of immune defense has resulted in a variety of strategies. Even unicellular organisms have rich arsenals of mechanisms for protection, such as restriction endonucleases, antimicrobial peptides, and RNA interference. In multicellular organisms, specialized immune cells have evolved, capable of recognition, phagocytosis, and killing of foreign cells as well as removing their own cells changed by damage, senescence, infection, or cancer. Additional humoral factors, such as the complement cascade, have developed that co-operate with cellular immunity in fighting infection and maintaining homeostasis. Defensive mechanisms based on germline-encoded receptors constitute a system known as innate immunity. In jaw vertebrates, this system is supplemented with a second system, adaptive immunity, which in contrast to innate immunity is based on diversification of immune receptors and on immunological memory in each individual.Usually, each newly evolved defense mechanism did not replace the previous one, but supplemented it, resulting in a layered structure of the immune system. The immune system is not one system but rather a sophisticated network of various defensive mechanisms operating on different levels, ranging from mechanisms common for every cell in the body to specialized immune cells and responses at the level of the whole organism. Adaptive changes in pathogens have shaped the evolution of the immune system at all levels.

Analysis of recombination signal sequences in zebrafish.

Recombination signal sequences (RSS) from immunoglobulin and TCRalpha genes of zebrafish were analyzed in comparison with RSS from human and species-specific features were revealed. In contrast to human RSS, in zebrafish RSS from both V(H) and TCRalpha genes the last nonamer position is not conserved. On the contrary, the fourth nonamer position, which is not conserved in human or mouse is conserved in zebrafish. The 12 bp spacers from human and zebrafish RSS contain 9 bp motif resembling nonamer sequence. Spacers in zebrafish 23 bp RSS from both immunoglobulins and TCRalpha contain 7 bp motif also resembling nonamer sequence while corresponding human sequences do not contain analogous motif. RSS are recognized by RAG1 protein, which also has specific features in teleost suggesting co-evolution of RAG1 with corresponding RSS.

The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z.

The only immunoglobulin heavy-chain classes known so far in teleosts have been mu and delta. We identify here a previously unknown class, immunoglobulin zeta, expressed in zebrafish and other teleosts. In the zebrafish heavy-chain locus, variable (V) gene segments lie upstream of two tandem diversity, joining and constant (DJC) clusters, resembling the mouse T cell receptor alpha (Tcra) and delta (Tcrd) locus. V genes rearrange to (DJC)(zeta) or to (DJC)(mu) without evidence of switch rearrangement. The zebrafish immunoglobulin zeta gene (ighz) and mouse Tcrd, which are proximal to the V gene array, are expressed earlier in development. In adults, ighz was expressed only in kidney and thymus, which are primary lymphoid organs in teleosts. This additional class adds complexity to the immunoglobulin repertoire and raises questions concerning the evolution of immunoglobulins and the regulation of the differential expression of ighz and ighm.

Expression of the winged helix/forkhead gene, foxn4, during zebrafish development.

The winged-helix/forkhead transcription factor gene, foxn4, is expressed in the nervous system of developing and adult zebrafish. Prominent expression sites include the olfactory placode, the basal layer of the olfactory epithelium, the neuroepithelium of the developing retina, the germinal zone of the differentiated eye, regions of motoneuron development in the neural tube and periventricular regions of the brain. The adult thymus is the only major site of foxn4 expression outside of the nervous system.

T cells and the thymus in developing zebrafish.

The expression of genes encoding T cell receptor (TCR) alpha was used to follow the development of the thymus and to analyze the distribution of T cells in zebrafish. In the thymus, expression was first detected, by in situ hybridization, at four days post fertilization. In RNA extracted from whole fish, TCRalpha transcripts were also detected at four days and reached adult levels at three weeks. At six weeks, TCRalpha was expressed throughout the thymus, whereas rag1 expression was localized to the peripheral regions. Expression of TCRalpha outside the thymus was detected at nine days. In adult peripheral organs, the greatest expression was in the pronephros, mesonephros and intestine; expression in the spleen became greater as fish age. Three new, relatively highly expressed, TCR Valpha families were identified.

Melatonin stimulates cell proliferation in zebrafish embryo and accelerates its development.

All vertebrates show a dramatic circadian rhythm in circulating melatonin with high levels at night and very low levels during daytime. In adults, melatonin is thought to synchronize other circadian rhythms and regulate seasonal rhythms in photoperiodic animals by acting on specific G-protein coupled receptors. The role of melatonin in development is unknown, even though melatonin receptors appear to be more highly expressed in developing embryos and neonates than in adults. In this study on zebrafish embryos, we describe a role for melatonin in increasing cell proliferation and accelerating development. We propose that melatonin has a role in extending the safe limit of proliferation rate at night to allow more rapid development when potentially damaging ultraviolet light is absent.

B cells develop in the zebrafish pancreas.

The zebrafish, with its transparent free-living embryo, is a useful organism for investigating early stages in lymphopoiesis. Previously, we showed that T cells differentiate in the thymus by day 4, but no sites for B cell differentiation were seen until 3 weeks. We report here that on day 4, we detect rearrangements of genes encoding B cell receptors in DNA extracted from whole fish. Also by day 4, rag1 transcripts are seen in the pancreas, an organ not previously associated with lymphopoiesis; by day 10, Igmu transcripts are detected here. Thus, in zebrafish, the pancreas assumes the role of both the liver in fetal mice and the spleen in neonatal mice.