Cellular self-organization: An overdrive in Cambrian diversity?

During the early Cambrian period metazoan life forms diverged at an accelerated rate to occupy multiple ecological niches on earth. A variety of explanations have been proposed to address this major evolutionary phenomenon termed the "Cambrian explosion." While most hypotheses address environmental, developmental, and ecological factors that facilitated evolutionary innovations, the biological basis for accelerated emergence of species diversity in the Cambrian period remains largely conjectural. Herein, we posit that morphogenesis by self-organization enables the uncoupling of genomic mutational landscape from phenotypic diversification. Evidence is provided for a two-tiered interpretation of genomic changes in metazoan animals wherein mutations not only impact upon function of individual cells, but also alter the self-organization outcome during developmental morphogenesis. We provide evidence that the morphological impacts of mutations on self-organization could remain repressed if associated with an unmet negative energetic cost. We posit that accelerated morphological diversification in transition to the Cambrian period has occurred by emergence of dormant (i.e., reserved) morphological novelties whose molecular underpinnings were seeded in the Precambrian period.

The fate of notch-1 transcript is linked to cell cycle dynamics by activity of a natural antisense transcript.

A core imprint of metazoan life is that perturbations of cell cycle are offset by compensatory changes in successive cellular generations. This trait enhances robustness of multicellular growth and requires transmission of signaling cues within a cell lineage. Notably, the identity and mode of activity of transgenerational signals remain largely unknown. Here we report the discovery of a natural antisense transcript encoded in exon 25 of notch-1 locus (nAS25) by which mother cells control the fate of notch-1 transcript in daughter cells to buffer against perturbations of cell cycle. The antisense transcript is transcribed at G1 phase of cell cycle from a bi-directional E2F1-dependent promoter in the mother cell where the titer of nAS25 is calibrated to the length of G1. Transmission of the antisense transcript from mother to daughter cells stabilizes notch-1 sense transcript in G0 phase of daughter cells by masking it from RNA editing and resultant nonsense-mediated degradation. In consequence, nAS25-mediated amplification of notch-1 signaling reprograms G1 phase in daughter cells to compensate for the altered dynamics of the mother cell. The function of nAS25/notch-1 in integrating G1 phase history of the mother cell into that of daughter cells is compatible with the predicted activity of a molecular oscillator, slower than cyclins, that coordinates cell cycle within cell lineage.

Notch ankyrin domain: evolutionary rise of a thermodynamic sensor.

Notch signalling pathway plays a key role in metazoan biology by contributing to resolution of binary decisions in the life cycle of cells during development. Outcomes such as proliferation/differentiation dichotomy are resolved by transcriptional remodelling that follows a switch from Notchon to Notchoff state, characterised by dissociation of Notch intracellular domain (NICD) from DNA-bound RBPJ. Here we provide evidence that transitioning to the Notchoff state is regulated by heat flux, a phenomenon that aligns resolution of fate dichotomies to mitochondrial activity. A combination of phylogenetic analysis and computational biochemistry was utilised to disclose structural adaptations of Notch1 ankyrin domain that enabled function as a sensor of heat flux. We then employed DNA-based micro-thermography to measure heat flux during brain development, followed by analysis in vitro of the temperature-dependent behaviour of Notch1 in mouse neural progenitor cells. The structural capacity of NICD to operate as a thermodynamic sensor in metazoans stems from characteristic enrichment of charged acidic amino acids in ß-hairpins of the ankyrin domain that amplify destabilising inter-residue electrostatic interactions and render the domain thermolabile. The instability emerges upon mitochondrial activity which raises the perinuclear and nuclear temperatures to 50 °C and 39 °C, respectively, leading to destabilization of Notch1 transcriptional complex and transitioning to the Notchoff state. Notch1 functions a metazoan thermodynamic sensor that is switched on by intercellular contacts, inputs heat flux as a proxy for mitochondrial activity in the Notchon state via the ankyrin domain and is eventually switched off in a temperature-dependent manner. Video abstract.

Notch pathway: a bistable inducer of biological noise?

Notch signalling pathway is central to development of metazoans. The pathway codes a binary fate switch. Upon activation, downstream signals contribute to resolution of fate dichotomies such as proliferation/differentiation or sub-lineage differentiation outcome. There is, however, an interesting paradox in the Notch signalling pathway. Despite remarkable predictability of fate outcomes instructed by the Notch pathway, the associated transcriptome is versatile and plastic. This inconsistency suggests the presence of an interface that compiles input from the plastic transcriptome of the Notch pathway but communicates only a binary output in biological decisions. Herein, we address the interface that determines fate outcomes. We provide an alternative hypothesis for the Notch pathway as a biological master switch that operates by induction of genetic noise and bistability in order to facilitate resolution of dichotomous fate outcomes in development.

Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.

Redox-Mediated Rewiring of Signalling Pathways: The Role of a Cellular Clock in Brain Health and Disease.

Metazoan signalling pathways can be rewired to dampen or amplify the rate of events, such as those that occur in development and aging. Given that a linear network topology restricts the capacity to rewire signalling pathways, such scalability of the pace of biological events suggests the existence of programmable non-linear elements in the underlying signalling pathways. Here, we review the network topology of key signalling pathways with a focus on redox-sensitive proteins, including PTEN and Ras GTPase, that reshape the connectivity profile of signalling pathways in response to an altered redox state. While this network-level impact of redox is achieved by the modulation of individual redox-sensitive proteins, it is the population by these proteins of critical nodes in a network topology of signal transduction pathways that amplifies the impact of redox-mediated reprogramming. We propose that redox-mediated rewiring is essential to regulate the rate of transmission of biological signals, giving rise to a programmable cellular clock that orchestrates the pace of biological phenomena such as development and aging. We further review the evidence that an aberrant redox-mediated modulation of output of the cellular clock contributes to the emergence of pathological conditions affecting the human brain.

Neural pericytes: A genomic archival state programmed by CHromatin topology.

Accumulating evidence suggests that mural pericytes, apart from stabilizing the associated microvessels, play additional roles in regeneration of local cellular elements. Herein, the mechanistic basis for such diverse and at times contradictory roles adopted by pericytes in the brain is reviewed. Core concepts of an emerging model are discussed wherein mural pericytes reside in a metastable "archival state" that conceals a neural progenitor identity. Upon angiogenic remodeling, a selected subpopulation of pericytes reclaim the progenitor state during transdifferentiation and contribute to neural regeneration. The genomic basis for neural transdifferentiation of pericytes is reviewed with reference to the extant literature.

Adaptive calcified matrix response of dental pulp to bacterial invasion is associated with establishment of a network of glial fibrillary acidic protein+/glutamine synthetase+ cells.

We report evidence for anatomical and functional changes of dental pulp in response to bacterial invasion through dentin that parallel responses to noxious stimuli reported in neural crest-derived sensory tissues. Sections of resin-embedded carious adult molar teeth were prepared for immunohistochemistry, in situ hybridization, ultrastructural analysis, and microdissection to extract mRNA for quantitative analyses. In odontoblasts adjacent to the leading edge of bacterial invasion in carious teeth, expression levels of the gene encoding dentin sialo-protein were 16-fold greater than in odontoblasts of healthy teeth, reducing progressively with distance from this site of the carious lesion. In contrast, gene expression for dentin matrix protein-1 by odontoblasts was completely suppressed in carious teeth relative to healthy teeth. These changes in gene expression were related to a gradient of deposited reactionary dentin that displayed a highly modified structure. In carious teeth, interodontoblastic dentin sialo-protein(-) cells expressing glutamine synthetase (GS) showed up-regulation of glial fibrillary acidic protein (GFAP). These cells extended processes that associated with odontoblasts. Furthermore, connexin 43 established a linkage between adjacent GFAP(+)/GS(+) cells in carious teeth only. These findings indicate an adaptive pulpal response to encroaching caries that includes the deposition of modified, calcified, dentin matrix associated with networks of GFAP(+)/GS(+) interodontoblastic cells. A regulatory role for the networks of GFAP(+)/GS(+) cells is proposed, mediated by the secretion of glutamate to modulate odontoblastic response.

Programmed genomic instability regulates neural transdifferentiation of human brain microvascular pericytes.

BACKGROUND: Transdifferentiation describes transformation in vivo of specialized cells from one lineage into another. While there is extensive literature on forced induction of lineage reprogramming in vitro, endogenous mechanisms that govern transdifferentiation remain largely unknown. The observation that human microvascular pericytes transdifferentiate into neurons provided an opportunity to explore the endogenous molecular basis for lineage reprogramming. RESULTS: We show that abrupt destabilization of the higher-order chromatin topology that chaperones lineage memory of pericytes is driven by transient global transcriptional arrest. This leads within minutes to localized decompression of the repressed competing higher-order chromatin topology and expression of pro-neural genes. Transition to neural lineage is completed by probabilistic induction of R-loops in key myogenic loci upon re-initiation of RNA polymerase activity, leading to depletion of the myogenic transcriptome and emergence of the neurogenic transcriptome. CONCLUSIONS: These findings suggest that the global transcriptional landscape not only shapes the functional cellular identity of pericytes, but also stabilizes lineage memory by silencing the competing neural program within a repressed chromatin state.

Cannibalized erythroblasts accelerate developmental neurogenesis by regulating mitochondrial dynamics.

Metabolic support was long considered to be the only developmental function of hematopoiesis, a view that is gradually changing. Here, we disclose a mechanism triggered during neurulation that programs brain development by donation of sacrificial yolk sac erythroblasts to neuroepithelial cells. At embryonic day (E) 8.5, neuroepithelial cells transiently integrate with the endothelium of yolk sac blood vessels and cannibalize intravascular erythroblasts as transient heme-rich endosymbionts. This cannibalistic behavior instructs precocious neuronal differentiation of neuroepithelial cells in the proximity of blood vessels. By experiments in vitro, we show that access to erythroblastic heme accelerates the pace of neurogenesis by induction of a truncated neurogenic differentiation program from a poised state. Mechanistically, the poised state is invoked by activation of the mitochondrial electron transport chain that leads to amplified production of reactive oxygen species in addition to omnipresent guanosine triphosphate (GTP) with consequential upregulation of pro-differentiation ß-catenin.

Genomic competition for noise reduction shaped evolutionary landscape of mir-4673.

The genomic platform that informs evolution of microRNA cascades remains unknown. Here we capitalised on the recent evolutionary trajectory of hominin-specific miRNA-4673, encoded in intron 4 of notch-1, to uncover the identity of one such precursor genomic element and the selective forces acting upon it. The miRNA targets genes that regulate Wnt/ß-catenin signalling cascade. Primary sequence of the microRNA and its target region in Wnt modulating genes evolved from homologous signatures mapped to homotypic cis-clusters recognised by TCF3/4 and TFAP2A/B/C families. Integration of homologous TFAP2A/B/C cis-clusters (short range inhibitor of ß-catenin) into the transcriptional landscape of Wnt cascade genes can reduce noise in gene expression. Probabilistic adoption of miRNA secondary structure by one such cis-signature in notch-1 reflected selection for superhelical curvature symmetry of precursor DNA to localise a nucleosome that overlapped the latter cis-cluster. By replicating the cis-cluster signature, non-random interactions of the miRNA with key Wnt modulator genes expanded the transcriptional noise buffering capacity via a coherent feed-forward loop mechanism. In consequence, an autonomous transcriptional noise dampener (the cis-cluster/nucleosome) evolved into a post-transcriptional one (the miRNA). The findings suggest a latent potential for remodelling of transcriptional landscape by miRNAs that capitalise on non-random distribution of genomic cis-signatures.

Coupled cycling and regulation of metazoan morphogenesis.

Metazoan animals are characterized by restricted phenotypic heterogeneity (i.e. morphological disparity) of organisms within various species, a feature that contrasts sharply with intra-species morphological diversity observed in the plant kingdom. Robust emergence of morphogenic blueprint in metazoan animals reflects restricted autonomy of individual cells in adoption of fate outcomes such as differentiation. Fates of individual cells are linked to and influenced by fates of neighboring cells at the population level. Such coupling is a common property of all self-organising systems and propels emergence of order from simple interactions between individual cells without supervision by external directing forces. As a consequence of coupling, expected functional relationship between the constituent cells of an organ system is robustly established concurrent with multiple rounds of cell division during morphogenesis. Notably, the molecular regulation of multicellular coupling during morphogenic self-organisation remains largely unexplored. Here, we review the existing literature on multicellular self-organisation with particular emphasis on recent discovery that ß-catenin is the key coupling factor that programs emergence of multi-cellular self-organisation by regulating synchronised cycling of individual cells.

Evolution of Resistance in Cancer: A Cell Cycle Perspective.

Resistance of neoplastic cells to therapy is considered a key challenge in the treatment of cancer. Emergence of resistance is commonly attributed to the gradual mutational evolution of neoplastic cells. However, accumulating evidence suggests that exogenous stressors could significantly accelerate the emergence of resistant clones during the course of treatment. Herein, we review molecular mechanisms that regulate the evolution of resistance in a tumor with particular emphasis on the role of cell cycle.

Coupled cycling programs multicellular self-organization of neural progenitors.

Self-organization is central to the morphogenesis of multicellular organisms. However, the molecular platform that coordinates the robust emergence of complex morphological patterns from local interactions between cells remains unresolved. Here we demonstrate that neural self- organization is driven by coupled cycling of progenitor cells. In a coupled cycling mode, intercellular contacts relay extrinsic cues to override the intrinsic cycling rhythm of an individual cell and synchronize the population. The stringency of coupling and hence the synchronicity of the population is programmed by recruitment of a key coupler, ß-catenin, into junctional complexes. As such, multicellular self-organization is driven by the same basic mathematical principle that governs synchronized behavior of macro-scale biological systems as diverse as the synchronized chirping of crickets, flashing of fireflies and schooling of fish; that is synchronization by coupling. It is proposed that coupled cycling foreshadows a fundamental adaptive change that facilitated evolution and diversification of multicellular life forms.

ß-Catenin: A Metazoan Filter for Biological Noise?

Molecular noise refers to fluctuations of biological signals that facilitate phenotypic heterogeneity in a population. While endogenous mechanisms exist to limit genetic noise in biological systems, such restrictions are sometimes removed to propel phenotypic variability as an adaptive strategy. Herein, we review evidence for the potential role of ß-catenin in restricting gene expression noise by transcriptional and post-transcriptional mechanisms. We discuss mechanisms that restrict intrinsic noise subsequent to nuclear mobilization of ß-catenin. Nuclear ß-catenin promotes initiation of transcription but buffers against the resultant noise by restraining transcription elongation. Acceleration of cell cycle, mediated via Wnt/ß-catenin downstream signals, further diminishes intrinsic noise by curtailing the efficiency of protein synthesis. Extrinsic noise, on the other hand, is restricted by ß-catenin-mediated regulation of major cellular stress pathways.

Neural microvascular pericytes contribute to human adult neurogenesis.

Consistent adult neurogenic activity in humans is observed in specific niches within the central nervous system. However, the notion of an adult neurogenic niche is challenged by accumulating evidence for ectopic neurogenic activity in other cerebral locations. Herein we interface precision of ultrastructural resolution and anatomical simplicity of accessible human dental pulp neurogenic zone to address this conflict. We disclose a basal level of adult neurogenic activity characterized by glial invasion of terminal microvasculature followed by release of individual platelet-derived growth factor receptor-ß mural pericytes and subsequent reprogramming into NeuN+ local interneurons. Concomitant angiogenesis, a signature of adult neurogenic niches, accelerates the rate of neurogenesis by amplifying release and proliferation of the mural pericyte population by ≈10-fold. Subsequent in vitro and in vivo experiments confirmed gliogenic and neurogenic capacities of human neural pericytes. Findings foreshadow the bimodal nature of the glio-vascular assembly where pericytes, under instruction from glial cells, can stabilize the quiescent microvasculature or enrich local neuronal microcircuits upon differentiation.

Neuroprotective effects of high-dose vs low-dose melatonin after blunt sciatic nerve injury.

INTRODUCTION: Melatonin, the secretory product of the pineal gland, has potent antioxidant properties. The aim of this study was to compare the effects of low-dose (10 mg/kg) vs high-dose (50 mg/kg) melatonin on early lipid peroxidation levels and ultrastructural changes in experimental blunt sciatic nerve injury (SNI). We believe this to be the first study to assess the dose-dependent neuroprotective effects of melatonin after a blunt peripheral nerve injury. MATERIALS AND METHODS: Rats were randomly allocated into 5 groups of 10 animals each. The SNI only rats underwent a nerve injury procedure. The SNI plus vehicle group received SNI and intraperitoneal injection of vehicle (diluted ethanol) as a placebo. The SNI plus low-dose or high-dose melatonin groups received intraperitoneal melatonin at doses of 10 mg/kg or 50 mg/kg, respectively. Controls had no operation, melatonin or vehicle injection. SNI was induced by clamping the sciatic nerve at the upper border of the quadratus femoris for 2 min. RESULTS: Sciatic nerve samples were harvested 6 h after nerve injury and processed for biochemical and ultrastructural analysis. Trauma increased the lipid peroxidation of the sciatic nerve by 3.6-fold (153.85 +/- 18.73 in SNI only vs 41.73 +/- 2.23 in control rats, P < 0.01). Low (P = 0.02) and high (P < 0.01) doses of melatonin attenuated the nerve lipid peroxidation by 25% and 57.25%, respectively (65.76 +/- 2.47 in high-dose vs 115.08 +/- 7.03 in low-dose melatonin groups). DISCUSSION: Although low-dose melatonin reduced trauma-induced myelin breakdown and axonal changes in the sciatic nerve, high-dose melatonin almost entirely neutralized any ultrastructural changes. CONCLUSION: Our results suggest that melatonin, especially at a dose of 50 mg/kg, has a potent neuroprotective effect and can preserve peripheral neural fibers from lipid peroxidative damage after blunt trauma. With further investigations, we hope that these data may prove useful to clinicians who treat patients with nerve injuries.

Study of root canal accessibility in human primary molars.

The aim of the present study was to provide a general scheme for pulpectomy of primary molars that may be useful for decision-making about negotiation of root canals and selection of appropriate instruments. A total of 160 vital primary molars in 85 patients (40 males, 45 females) aged 4-6 years were selected. After taking primary radiographs, local anesthesia was induced, and the teeth were isolated using a rubber dam. Canal accessibility index (CAI) and tooth accessibility index (TAI) were calculated according to initial file size. Mandibular first molars had either three canals (79.2%) or four canals (20.8%), and all second molars had four canals. Maxillary first molars had three canals and second molars had either three canals (70.9%) or four canals (29.1%). Lower accessibility of the mandibular first molar distobuccal root accounted for the lower accessibility of these teeth in comparison with mandibular second molars. While three-canal maxillary second molars were more accessible due to the lower accessibility of the distobuccal canal of the maxillary first molar, poor accessibility of the distal canal in four-canal second molars was responsible for the difficult accessibility of these teeth. In conclusion, it seems that the accessibility of a single canal in each tooth determines the difficulty of accessibility for any given tooth. Moreover, while primary second molars are more accessible than first molars, all of them are negotiable.

miR4673 improves fitness profile of neoplastic cells by induction of autophagy.

Therapeutic resistance of neoplasms is mainly attributed to gradual evolution of mutational profile1. Here, we demonstrate a microRNA-mediated mechanism that effectively improves fitness of SKBR3 mammary carcinoma cells by cytoplasmic reprogramming. The reprogramming is triggered by endogenous miR4673 transcribed from notch-1 locus. The miRNA downregulates cdk-18, a cyclin-dependent kinase that regulates M-G1 transition in cycling cells2,3. Suppression of cdk-18 triggers mitophagy and autophagy. Due to high autophagic flux, oestrogen receptor-1+/progesterone receptor+/p53+ (Esr1+/Pr+/p53+) SKBR3 cells are coerced into an Esr1-/Prlow/p53-profile. Increased mitophagy in combination with proteasomal degradation of p53 transiently arrests the cycling cells at G0 and enhances radio-resistance of the SKBR3 population. These findings highlight the impact on cancer therapy of non-encoded neoplastic resistance, arising as a consequence of miRNA-mediated autophagic reprogramming that uncouples phenotype and genotype.

A method for investigating the cellular response to cyclic tension or compression in three-dimensional culture.

We have an interest in the cellular response to mechanical stimuli, and here describe an in-vitro method to examine the response of cells cultured in a three-dimensional matrix to mechanical compressive and tensile stress. Synthetic aliphatic polyester scaffolds coated with 45S5 bioactive glass were seeded with human dental follicular cells (HDFC), and attached to well inserts and magnetic endplates in six well palates. Scaffolds were subjected to either cyclic 10% tensile deformation, or 8% compression, at 1 Hz and 2 Hz respectively for 6, 24 or 48 h, by uniaxial motion of magnetically-coupled endplates. It was possible to isolate high quality mRNA from cells in these scaffolds, as demonstrated by high RNA integrity numbers scores, and ability to perform meaningful cRNA microarray analysis, in which 669 and 727 genes were consistently upregulated, and 662 and 518 genes down regulated at all times studied under tensile and compressive loading conditions respectively. MetaCore analysis revealed the most regulated gene ontogenies under both loading conditions to be for: cytoskeletal remodelling; cell adhesion-chemokines and adhesion; cytoskeleton remodelling-TGF WNT and cytoskeletal remodelling pathways. We believe the method here described will be of value for analysis of the cellular response to cyclic loading.