A model of the PI cycle reveals the regulating roles of lipid-binding proteins and pitfalls of using mosaic biological data.

The phosphatidylinositol (PI) cycle is central to eukaryotic cell signaling. Its complexity, due to the number of reactions and lipid and inositol phosphate intermediates involved makes it difficult to analyze experimentally. Computational modelling approaches are seen as a way forward to elucidate complex biological regulatory mechanisms when this cannot be achieved solely through experimental approaches. Whilst mathematical modelling is well established in informing biological systems, many models are often informed by data sourced from multiple unrelated cell types (mosaic data) or from purified enzyme data. In this work, we develop a model of the PI cycle informed by experimental and omics data taken from a single cell type, namely platelets. We were able to make a number of predictions regarding the regulation of PI cycle enzymes, the importance of the number of receptors required for successful GPCR signaling and the importance of lipid- and protein-binding proteins in regulating second messenger outputs. We then consider how pathway behavior differs, when fully informed by data for HeLa cells and show that model predictions remain consistent. However, when informed by mosaic experimental data model predictions greatly vary illustrating the risks of using mosaic datasets from unrelated cell types.

Precise Quantification of Platelet Proteins and Their Phosphorylation States.

Systems biology and modeling approaches require quantitative data-rich temporal experiments to better understand the dynamics and regulation of the components of the signaling pathways that governs cell biology and physiology. Here we present a modified Western blotting method to rapidly analyze and accurately quantify protein copy number, and their respective phosphorylation states at specific sites over detailed time courses.

Regulation of Early Steps of GPVI Signal Transduction by Phosphatases: A Systems Biology Approach.

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation.

The Fox genes of Branchiostoma floridae.

The Fox genes are united by encoding a fork head domain, a deoxyribonucleic acid (DNA)-binding domain of the winged-helix type that marks these genes as encoding transcription factors. Vertebrate Fox genes are classified into 23 subclasses named from FoxA to FoxS. We have surveyed the genome of the amphioxus Branchiostoma floridae, identifying 32 distinct Fox genes representing 21 of these 23 subclasses. The missing subclasses, FoxR and FoxS, are specific to vertebrates, and in addition, B. floridae has one further group, FoxAB, that is not found in vertebrates. Hence, we conclude B. floridae has maintained a high level of Fox gene diversity. Expressed sequence tag and complementary DNA sequence data support the expression of 23 genes. Several linkages between B. floridae Fox genes were noted, including some that have evolved relatively recently via tandem duplication in the amphioxus lineage and others that are more ancient.

Expression of FoxC, FoxF, FoxL1, and FoxQ1 genes in the dogfish Scyliorhinus canicula defines ancient and derived roles for Fox genes in vertebrate development.

In the human genome, members of the FoxC, FoxF, FoxL1, and FoxQ1 gene families are found in two paralagous clusters. Here we characterize all four gene families in the dogfish Scyliorhinus canicula, a member of the cartilaginous fish lineage that diverged before the radiation of osteichthyan vertebrates. We identify two FoxC genes, two FoxF genes, and single FoxQ1 and FoxL1 genes, demonstrating cluster duplication preceded the radiation of gnathostomes. The expression of all six genes was analyzed by in situ hybridization. The results show conserved expression of FoxL1, FoxF, and FoxC genes in different compartments of the mesoderm and of FoxQ1 in pharyngeal endoderm and its derivatives, confirming these as ancient sites of Fox gene expression, and also illustrate multiple cases of lineage-specific expression domains. Comparison to invertebrate chordates shows that the majority of conserved vertebrate expression domains mark tissues that are part of the primitive chordate body plan.

Restricted expression of NADPH oxidase/peroxidase gene (Duox) in zone VII of the ascidian endostyle.

The ascidian Ciona intestinalis, a marine invertebrate chordate, is an emerging model system for developmental and evolutionary studies. The endostyle, one of the characteristic organs of ascidians, is a pharyngeal structure with iodine-concentrating and peroxidase activities and is therefore considered to be homologous to the follicular thyroid of higher vertebrates. We have previously reported that a limited part of the endostyle (zone VII) is marked by the expression of orthologs of the thyroid peroxidase (TPO) and thyroid transcription factor-2 (TTF-2/FoxE) genes. In this study, we have identified the Ciona homolog of NADPH oxidase/peroxidase (Duox), which provides hydrogen peroxide (H(2)O(2)) for iodine metabolism by TPO in the vertebrate thyroid. Expression patterns assessed by in situ hybridization have revealed that Ciona Duox (Ci-Duox) is predominantly expressed in the dorsal part of zone VII of the endostyle. Furthermore, two-color fluorescent in situ hybridization with Ci-Duox and Ciona TPO (CiTPO) has revealed that the ventral boundary of the Ci-Duox domain of expression is more dorsal than that of CiTPO. We have also characterized several genes, such as Ci-Fgf8/17/18, 5HT7, and Ci-NK4, which are predominantly expressed in the ventral part of zone VII, in a region complementary to the Ci-Duox expression domain. These observations suggest that, at the molecular level, zone VII has a complex organization that might have some impact on the specification of cell types and functions in this thyroid-equivalent element of the ascidian endostyle.

The evolution of sensory placodes.

The vertebrate cranial sensory placodes are ectodermal embryonic patches that give rise to sensory receptor cells of the peripheral paired sense organs and to neurons in the cranial sensory ganglia. Their differentiation and the genetic pathways that underlay their development are now well understood. Their evolutionary history, however, has remained obscure. Recent molecular work, performed on close relatives of the vertebrates, demonstrated that some sensory placodes (namely the adenohypophysis, the olfactory, and accoustico-lateralis placodes) first evolved at the base of the chordate lineage, while others might be specific to vertebrates. Combined with morphological and cellular fate data, these results also suggest that the sensory placodes of the ancestor of all chordates differentiated into a wide range of structures, most likely to fit the lifestyle and environment of each species.

Molecular evidence from ascidians for the evolutionary origin of vertebrate cranial sensory placodes.

Cranial sensory placodes are specialised areas of the head ectoderm of vertebrate embryos that contribute to the formation of the cranial sense organs and associated ganglia. Placodes are often considered a vertebrate innovation, and their evolution has been hypothesised as one key adaptation underlying the evolution of active predation by primitive vertebrates. Here, we review recent molecular evidence pertinent to understanding the evolutionary origin of placodes. The development of vertebrate placodes is regulated by numerous genes, including members of the Pax, Six, Eya, Fox, Phox, Neurogenin and Pou gene families. In the sea squirt Ciona intestinalis (a basal chordate and close relative of the vertebrates), orthologues of these genes are deployed in the development of the oral and atrial siphons, structures used for filter feeding by the sessile adult. Our interpretation of these findings is that vertebrate placodes and sea squirt siphon primordia have evolved from the same patches of specialised ectoderm present in the common ancestor of the chordates.

Molecular evidence from Ciona intestinalis for the evolutionary origin of vertebrate sensory placodes.

Cranial sensory placodes are focused areas of the head ectoderm of vertebrates that contribute to the development of the cranial sense organs and their associated ganglia. Placodes have long been considered a key character of vertebrates, and their evolution is proposed to have been essential for the evolution of an active predatory lifestyle by early vertebrates. Despite their importance for understanding vertebrate origins, the evolutionary origin of placodes has remained obscure. Here, we use a panel of molecular markers from the Six, Eya, Pax, Dach, FoxI, COE and POUIV gene families to examine the tunicate Ciona intestinalis for evidence of structures homologous to vertebrate placodes. Our results identify two domains of Ciona ectoderm that are marked by the genetic cascade that regulates vertebrate placode formation. The first is just anterior to the brain, and we suggest this territory is equivalent to the olfactory/adenohypophyseal placodes of vertebrates. The second is a bilateral domain adjacent to the posterior brain and includes cells fated to form the atrium and atrial siphon of adult Ciona. We show this bares most similarity to placodes fated to form the vertebrate acoustico-lateralis system. We interpret these data as support for the hypothesis that sensory placodes did not arise de novo in vertebrates, but evolved from pre-existing specialised areas of ectoderm that contributed to sensory organs in the common ancestor of vertebrates and tunicates.

Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates.

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/FoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage.

The amphioxus FoxQ1 gene is expressed in the developing endostyle.

The FoxQ1 genes form a distinct group within the Fox (also known as forkhead) gene family. We have isolated a gene from the amphioxus Branchiostoma floridae that encodes a forkhead domain with high identity to FoxQ1 genes in other chordates. Molecular phylogenetic analysis places AmphiFoxQ1 in a robust grouping with vertebrate FoxQ1 genes and with Ciona intestinalis Ci-FoxQ1. This group is separate from that containing AmphiFoxQ2, which instead groups with other invertebrate Fox genes. The expression of AmphiFoxQ1 was analysed by whole mount in situ hybridisation. The results show that AmphiFoxQ1 expression is confined to the developing endoderm, and specifically marks the endostyle and associated peripharyngeal bands of amphioxus larvae. Ci-FoxQ1 is also expressed in the endostyle, highlighting this as a conserved site of FoxQ1 gene expression in basal chordates.

Expression of AmphiCoe, an amphioxus COE/EBF gene, in the developing central nervous system and epidermal sensory neurons.

The COE/EBF gene family marks a subset of prospective neurons in the vertebrate central and peripheral nervous system, including neurons deriving from some ectodermal placodes. Since placodes are often considered unique to vertebrates, we have characterised an amphioxus COE/EBF gene with the aim of using it as a marker to examine the timing and location of peripheral neuron differentiation. A single COE/EBF family member, AmphiCoe, was isolated from the amphioxus Branchiostoma floridae. AmphiCoe lies basal to the vertebrate COE/EBF genes in molecular phylogenetic analysis, suggesting that the duplications that formed the vertebrate COE/EBF family were specific to the vertebrate lineage. AmphiCoe is expressed in the central nervous system and in a small number of scattered ectodermal cells on the flanks of neurulae stage embryos. These cells become at least largely recessed beneath the ectoderm. Scanning electron microscopy was used to examine embryos in which the ectoderm had been partially peeled away. This revealed that these cells have neuronal morphology, and we infer that they are the precursors of epidermal primary sensory neurons. These characters lead us to suggest that differentiation of some ectodermal cells into sensory neurons with a tendency to sink beneath the embryonic surface represents a primitive feature that has become incorporated into placodes during vertebrate evolution.

Pax gene expression in the developing central nervous system of Ciona intestinalis.

We compare the expression patterns in Ciona intestinalis of three members of the Pax gene family, CiPax3/7, CiPax6 and Cipax2/5/8. All three genes are expressed in restricted patterns in the developing central nervous system. At the tailbud stage, CiPax3/7 is present in three patches in the brain and along the posterior neural tube, CiPax6 throughout the anterior brain and along the posterior neural tube and CiPax2/5/8 in a restricted region of the posterior brain. Double in situ hybridisations were used to identify areas of overlap between the expression of different genes. This showed that CiPax3/7 overlaps with the boundaries of CiPax6 expression in the anterior brain, and with CiPax2/5/8 in the posterior brain. The overlap between CiPax3/7 and CiPax2/5/8 is unlike that described in the ascidian Halocynthia rorezti.

Phylogenetic relationships of the Fox (Forkhead) gene family in the Bilateria.

The Forkhead or Fox gene family encodes putative transcription factors. There are at least four Fox genes in yeast, 16 in Drosophila melanogaster (Dm) and 42 in humans. Recently, vertebrate Fox genes have been classified into 17 groups named FoxA to FoxQ. Here, we extend this analysis to invertebrates, using available sequences from D. melanogaster, Anopheles gambiae (Ag), Caenorhabditis elegans (Ce), the sea squirt Ciona intestinalis (Ci) and amphioxus Branchiostoma floridae (Bf), from which we also cloned several Fox genes. Phylogenetic analyses lend support to the previous overall subclassification of vertebrate genes, but suggest that four subclasses (FoxJ, L, N and Q) could be further subdivided to reflect their relationships to invertebrate genes. We were unable to identify orthologs of Fox subclasses E, H, I, J, M and Q1 in D. melanogaster, A. gambiae or C. elegans, suggesting either considerable loss in ecdysozoans or the evolution of these subclasses in the deuterostome lineage. Our analyses suggest that the common ancestor of protostomes and deuterostomes had a minimum complement of 14 Fox genes.

Characterisation of an amphioxus Fringe gene and the evolution of the vertebrate segmentation clock.

In mouse and chick embryos, cyclic expression of lunatic fringe has an important role in the regulation of mesoderm segmentation. We have isolated a Fringe gene from the protochordate amphioxus. Amphioxus is the closest living relative of the vertebrates, and has mesoderm that is definitively segmented in a manner that is similar to, and probably homologous with, that of vertebrates. AmphiFringe is placed basal to vertebrate Fringe genes in molecular phylogenetic analyses, indicating that the duplications that formed radical-, manic- and lunatic fringe are specific to the vertebrate lineage. AmphiFringe expression was detected in the anterior neural plate of early neurulae, where it resolved into a series of segmental patches by the mid-neurulae stage. No AmphiFringe transcripts were detected in the mesoderm. Based on these observations, we propose a model depicting a successive recruitment of Fringe in the maintenance then regulation of segmentation during vertebrate evolution.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. III. Genes for Fox, ETS, nuclear receptors and NFkappaB.

A survey against the draft genome sequence and the cDNA/EST database of Ciona intestinalis identified a number of genes encoding transcription factors regulating a variety of processes including development. In the present study, we describe almost complete sets of genes for Fox, ETS-domain transcription factors, nuclear receptors, and NFkappaB as well as other factors regulating NFkappaB activity, with their phylogenetic nature. Vertebrate Fox transcription factors are currently delineated into 17 subfamilies: FoxA to FoxQ. The present survey yielded 29 genes of this family in the Ciona genome, 24 of which were Ciona orthologues of known Fox genes. In addition, we found 15 ETS genes, 17 nuclear receptor genes, and several NFkappaB signaling pathway genes in the Ciona genome. The number of Ciona genes in each family is much smaller than that of vertebrates, which represents a simplified feature of the ascidian genome. For example, humans have two NFkappaB genes, three Rel genes, and five NFAT genes, while Ciona has one gene for each family. The Ciona genome also contains smaller numbers of genes for the NFkappaB regulatory system, i.e. after the split of ascidians/vertebrates, vertebrates evolved a more complex NFkappaB system. The present results therefore provide molecular information for the investigation of complex developmental processes, and an insight into chordate evolution.

The evolution of chordate neural segmentation.

Amphioxus is the closest relative to vertebrates but lacks key vertebrate characters, like rhombomeres, neural crest cells, and the cartilaginous endoskeleton. This reflects major differences in the developmental patterning of neural and mesodermal structures between basal chordates and vertebrates. Here, we analyse the expression pattern of an amphioxus FoxB ortholog and an amphioxus single-minded ortholog to gain insight into the evolution of vertebrate neural segmentation. AmphiFoxB expression shows cryptic segmentation of the cerebral vesicle and hindbrain, suggesting that neuromeric segmentation of the chordate neural tube arose before the origin of the vertebrates. In the forebrain, AmphiFoxB expression combined with AmphiSim and other amphioxus gene expression patterns shows that the cerebral vesicle is divided into several distinct domains: we propose homology between these domains and the subdivided diencephalon and midbrain of vertebrates. In the Hox-expressing region of the amphioxus neural tube that is homologous to the vertebrate hindbrain, AmphiFoxB shows the presence of repeated blocks of cells along the anterior-posterior axis, each aligned with a somite. This and other data lead us to propose a model for the evolution of vertebrate rhombomeric segmentation, in which rhombomere evolution involved the transfer of mechanisms regulating neural segmentation from vertical induction by underlying segmented mesoderm to horizontal induction by graded retinoic acid signalling. A consequence of this would have been that segmentation of vertebrate head mesoderm would no longer have been required, paving the way for the evolution of the unsegmented head mesoderm seen in living vertebrates.

The Fox and the thyroid: the amphioxus perspective.

The evolutionary origins of several vertebrate organs are still controversial. The thyroid is classically thought to derive directly from the endostyle (a pharyngeal organ found in urochordates, cephalochordates and lampreys). Several molecular and biochemical lines of evidence agree with this scenario. However, a recent paper,1 describing the expression of a FoxE ortholog in amphioxus, suggests that some molecular pathways might actually have been recruited from an adjacent region of the pharynx. Although additional data from urochordates and lamprey are needed to confirm this hypothesis; these results propose an interesting new scenario for thyroid evolution that involved the reorganisation of genetical and morphological features in the pharyngeal endoderm in order to give rise to a entirely new organ. They also give an indication that the ancestral role of the FoxE gene family was probably limited to the differentiation of part of the pharynx.

Gene duplication and divergence in the early evolution of vertebrates.

The duplication-degeneration-complementation model of duplicate gene preservation by subfunctionalisation is currently the best explanation for the high level of retention of duplicate genes in early vertebrate evolution. But a direct test of the applicability of this model to such ancient evolutionary events may be difficult. More likely, recent duplications in other lineages will allow us to establish general principles concerning the fate of genes of different types that are duplicated in different ways. These principles may be then extrapolated to understanding the early evolution of the vertebrates.