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1.  Deconstructing pheromone-mediated behavior one layer at a time 
BMC Biology  2014;12:33.
The vomeronasal organ, a sensory structure within the nasal cavity of most tetrapods, detects pheromones that influence socio-sexual behavior. It has two neuronal layers, each patterned by distinct receptor sub-families coupled to different G-proteins. Work recently published in this journal found female mice with one layer genetically inactivated are deficient in a surprisingly wide range of reproductive behaviors, providing new insights into how the nose can influence the brain.
See research article:
PMCID: PMC4037717  PMID: 24884538
2.  Casposons: a new superfamily of self-synthesizing DNA transposons at the origin of prokaryotic CRISPR-Cas immunity 
BMC Biology  2014;12:36.
Diverse transposable elements are abundant in genomes of cellular organisms from all three domains of life. Although transposons are often regarded as junk DNA, a growing body of evidence indicates that they are behind some of the major evolutionary innovations. With the growth in the number and diversity of sequenced genomes, previously unnoticed mobile elements continue to be discovered.
We describe a new superfamily of archaeal and bacterial mobile elements which we denote casposons because they encode Cas1 endonuclease, a key enzyme of the CRISPR-Cas adaptive immunity systems of archaea and bacteria. The casposons share several features with self-synthesizing eukaryotic DNA transposons of the Polinton/Maverick class, including terminal inverted repeats and genes for B family DNA polymerases. However, unlike any other known mobile elements, the casposons are predicted to rely on Cas1 for integration and excision, via a mechanism similar to the integration of new spacers into CRISPR loci. We identify three distinct families of casposons that differ in their gene repertoires and evolutionary provenance of the DNA polymerases. Deep branching of the casposon-encoded endonuclease in the Cas1 phylogeny suggests that casposons played a pivotal role in the emergence of CRISPR-Cas immunity.
The casposons are a novel superfamily of mobile elements, the first family of putative self-synthesizing transposons discovered in prokaryotes. The likely contribution of capsosons to the evolution of CRISPR-Cas parallels the involvement of the RAG1 transposase in vertebrate immunoglobulin gene rearrangement, suggesting that recruitment of endonucleases from mobile elements as ready-made tools for genome manipulation is a general route of evolution of adaptive immunity.
PMCID: PMC4046053  PMID: 24884953
Mobile genetic elements; CRISPR-Cas system; Adaptive immunity; Transposons; Archaea; DNA polymerases
3.  Elongated TCR alpha chain CDR3 favors an altered CD4 cytokine profile 
BMC Biology  2014;12:32.
CD4 T lymphocyte activation requires T cell receptor (TCR) engagement by peptide/MHC (major histocompatibility complex) (pMHC). The TCR complementarity-determining region 3 (CDR3) contains variable α and β loops critical for pMHC recognition. During any immune response, tuning of TCR usage through progressive clonal selection occurs. Th1 and Th2 cells operate at different avidities for activation and display distinct transcriptional programs, although polarization may be plastic, influenced by pathogens and cytokines. We therefore hypothesized that CDR3αβ sequence features may intrinsically influence CD4 phenotype during progression of a response.
We show that CD4 polarization involves distinct CDR3α usage: Th1 and Th17 cells favored short TCR CDR3α sequences of 12 and 11 amino acids, respectively, while Th2 cells favored elongated CDR3α loops of 14 amino acids, with lower predicted affinity. The dominant Th2- and Th1-derived TCRα sequences with14 amino acid CDR3 loops and 12 amino acid CDR3 loops, respectively, were expressed in TCR transgenics. The functional impact of these TCRα transgenes was assessed after in vivo priming with a peptide/adjuvant. The short, Th1-derived receptor transgenic T cell lines made IFNγ, but not IL-4, 5 or 13, while the elongated, Th2-derived receptor transgenic T cell lines made little or no IFNγ, but increased IL-4, 5 and 13 with progressive re-stimulations, mirrored by GATA-3 up-regulation. T cells from primed Th2 TCRα transgenics selected dominant TCR Vβ expansions, allowing us to generate TCRαβ transgenics carrying the favored, Th2-derived receptor heterodimer. Primed T cells from TCRαβ transgenics made little or no IL-17 or IFNγ, but favored IL-9 after priming with Complete Freund’s adjuvant and IL-4, 5, 9, 10 and 13 after priming with incomplete Freund’s. In tetramer-binding studies, this transgenic receptor showed low binding avidity for pMHC and polarized T cell lines show TCR avidity for Th17 > Th1 > Th2. While transgenic expression of a Th2-derived, ‘elongated’ TCR-CDR3α and the TCRαβ pair, clearly generated a program shifted away from Th1 immunity and with low binding avidity, cytokine-skewing could be over-ridden by altering peptide challenge dose.
We propose that selection from responding clones with distinctive TCRs on the basis of functional avidity can direct a preference away from Th1 effector responses, favoring Th2 cytokines.
PMCID: PMC4046507  PMID: 24886643
CD4 T cell; T cell receptor; Cytokine; Transgenic; Mouse; Th17; Th1; Th2
4.  A wide range of pheromone-stimulated sexual and reproductive behaviors in female mice depend on G protein Gαo 
BMC Biology  2014;12:31.
Optimal reproductive fitness is essential for the biological success and survival of species. The vomeronasal organ is strongly implicated in the display of sexual and reproductive behaviors in female mice, yet the roles that apical and basal vomeronasal neuron populations play in controlling these gender-specific behaviors remain largely unclear.
To dissect the neural pathways underlying these functions, we genetically inactivated the basal vomeronasal organ layer using conditional, cell-specific ablation of the G protein Gαo. Female mice mutant for Gαo show severe alterations in sexual and reproductive behaviors, timing of puberty onset, and estrous cycle. These mutant mice are insensitive to reproductive facilitation stimulated by male pheromones that accelerate puberty and induce ovulation. Gαo-mutant females exhibit a striking reduction in sexual receptivity or lordosis behavior to males, but gender discrimination seems to be intact. These mice also show a loss in male scent preference, which requires a learned association for volatile olfactory signals with other nonvolatile ownership signals that are contained in the high molecular weight fraction of male urine. Thus, Gαo impacts on both instinctive and learned social responses to pheromones.
These results highlight that sensory neurons of the Gαo-expressing vomeronasal subsystem, together with the receptors they express and the molecular cues they detect, control a wide range of fundamental mating and reproductive behaviors in female mice.
PMCID: PMC4038847  PMID: 24886577
Bruce effect; Estrus induction; Gαo signaling; Lordosis; Mate recognition; Puberty acceleration; Reproduction
5.  Specific NuRD components are required for fin regeneration in zebrafish 
BMC Biology  2014;12:30.
Epimorphic regeneration of a missing appendage in fish and urodele amphibians involves the creation of a blastema, a heterogeneous pool of progenitor cells underneath the wound epidermis. Current evidence indicates that the blastema arises by dedifferentiation of stump tissues in the vicinity of the amputation. In response to tissue loss, silenced developmental programs are reactivated to form a near-perfect copy of the missing body part. However, the importance of chromatin regulation during epimorphic regeneration remains poorly understood.
We found that specific components of the Nucleosome Remodeling and Deacetylase complex (NuRD) are required for fin regeneration in zebrafish. Transcripts of the chromatin remodeler chd4a/Mi-2, the histone deacetylase hdac1/HDAC1/2, the retinoblastoma-binding protein rbb4/RBBP4/7, and the metastasis-associated antigen mta2/MTA were specifically co-induced in the blastema during adult and embryonic fin regeneration, and these transcripts displayed a similar spatial and temporal expression patterns. In addition, chemical inhibition of Hdac1 and morpholino-mediated knockdown of chd4a, mta2, and rbb4 impaired regenerative outgrowth, resulting in reduction in blastema cell proliferation and in differentiation defects.
Altogether, our data suggest that specialized NuRD components are induced in the blastema during fin regeneration and are involved in blastema cell proliferation and redifferentiation of osteoblast precursor cells. These results provide in vivo evidence for the involvement of key epigenetic factors in the cellular reprogramming processes occurring during epimorphic regeneration in zebrafish.
PMCID: PMC4038851  PMID: 24779377
NuRD; Blastema; Fin; Regeneration; Zebrafish
6.  Reprogramming of the chick retinal pigmented epithelium after retinal injury 
BMC Biology  2014;12:28.
One of the promises in regenerative medicine is to regenerate or replace damaged tissues. The embryonic chick can regenerate its retina by transdifferentiation of the retinal pigmented epithelium (RPE) and by activation of stem/progenitor cells present in the ciliary margin. These two ways of regeneration occur concomitantly when an external source of fibroblast growth factor 2 (FGF2) is present after injury (retinectomy). During the process of transdifferentiation, the RPE loses its pigmentation and is reprogrammed to become neuroepithelium, which differentiates to reconstitute the different cell types of the neural retina. Somatic mammalian cells can be reprogrammed to become induced pluripotent stem cells by ectopic expression of pluripotency-inducing factors such as Oct4, Sox2, Klf4, c-Myc and in some cases Nanog and Lin-28. However, there is limited information concerning the expression of these factors during natural regenerative processes. Organisms that are able to regenerate their organs could share similar mechanisms and factors with the reprogramming process of somatic cells. Herein, we investigate the expression of pluripotency-inducing factors in the RPE after retinectomy (injury) and during transdifferentiation in the presence of FGF2.
We present evidence that upon injury, the quiescent (p27Kip1+/BrdU-) RPE cells transiently dedifferentiate and express sox2, c-myc and klf4 along with eye field transcriptional factors and display a differential up-regulation of alternative splice variants of pax6. However, this transient process of dedifferentiation is not sustained unless FGF2 is present. We have identified lin-28 as a downstream target of FGF2 during the process of retina regeneration. Moreover, we show that overexpression of lin-28 after retinectomy was sufficient to induce transdifferentiation of the RPE in the absence of FGF2.
These findings delineate in detail the molecular changes that take place in the RPE during the process of transdifferentiation in the embryonic chick, and specifically identify Lin-28 as an important factor in this process. We propose a novel model in which injury signals initiate RPE dedifferentiation, while FGF2 up-regulates Lin-28, allowing for RPE transdifferentiation to proceed.
PMCID: PMC4026860  PMID: 24742279
Regeneration; Retina; Transdifferentiation
7.  Genomic composition and evolution of Aedes aegypti chromosomes revealed by the analysis of physically mapped supercontigs 
BMC Biology  2014;12:27.
An initial comparative genomic study of the malaria vector Anopheles gambiae and the yellow fever mosquito Aedes aegypti revealed striking differences in the genome assembly size and in the abundance of transposable elements between the two species. However, the chromosome arms homology between An. gambiae and Ae. aegypti, as well as the distribution of genes and repetitive elements in chromosomes of Ae. aegypti, remained largely unexplored because of the lack of a detailed physical genome map for the yellow fever mosquito.
Using a molecular landmark-guided fluorescent in situ hybridization approach, we mapped 624 Mb of the Ae. aegypti genome to mitotic chromosomes. We used this map to analyze the distribution of genes, tandem repeats and transposable elements along the chromosomes and to explore the patterns of chromosome homology and rearrangements between Ae. aegypti and An. gambiae. The study demonstrated that the q arm of the sex-determining chromosome 1 had the lowest gene content and the highest density of minisatellites. A comparative genomic analysis with An. gambiae determined that the previously proposed whole-arm synteny is not fully preserved; a number of pericentric inversions have occurred between the two species. The sex-determining chromosome 1 had a higher rate of genome rearrangements than observed in autosomes 2 and 3 of Ae. aegypti.
The study developed a physical map of 45% of the Ae. aegypti genome and provided new insights into genomic composition and evolution of Ae. aegypti chromosomes. Our data suggest that minisatellites rather than transposable elements played a major role in rapid evolution of chromosome 1 in the Aedes lineage. The research tools and information generated by this study contribute to a more complete understanding of the genome organization and evolution in mosquitoes.
PMCID: PMC4021624  PMID: 24731704
Physical mapping; Mosquito; Genome; Chromosome
8.  Q&A: What are strigolactones and why are they important to plants and soil microbes? 
BMC Biology  2014;12:19.
PMCID: PMC3994223  PMID: 24685292
9.  Evolutionary origin of gastrulation: insights from sponge development 
BMC Biology  2014;12:26.
The evolutionary origin of gastrulation—defined as a morphogenetic event that leads to the establishment of germ layers—remains a vexing question. Central to this debate is the evolutionary relationship between the cell layers of sponges (poriferans) and eumetazoan germ layers. Despite considerable attention, it remains unclear whether sponge cell layers undergo progressive fate determination akin to eumetazoan primary germ layer formation during gastrulation.
Here we show by cell-labelling experiments in the demosponge Amphimedon queenslandica that the cell layers established during embryogenesis have no relationship to the cell layers of the juvenile. In addition, juvenile epithelial cells can transdifferentiate into a range of cell types and move between cell layers. Despite the apparent lack of cell layer and fate determination and stability in this sponge, the transcription factor GATA, a highly conserved eumetazoan endomesodermal marker, is expressed consistently in the inner layer of A. queenslandica larvae and juveniles.
Our results are compatible with sponge cell layers not undergoing progressive fate determination and thus not being homologous to eumetazoan germ layers. Nonetheless, the expression of GATA in the sponge inner cell layer suggests a shared ancestry with the eumetazoan endomesoderm, and that the ancestral role of GATA in specifying internalised cells may antedate the origin of germ layers. Together, these results support germ layers and gastrulation evolving early in eumetazoan evolution from pre-existing developmental programs used for the simple patterning of cells in the first multicellular animals.
PMCID: PMC4021757  PMID: 24678663
Evolution; Gastrulation; Germ layers; Sponge development; GATA
10.  LIM homeodomain transcription factor Isl1 directs normal pyloric development by targeting Gata3 
BMC Biology  2014;12:25.
Abnormalities in pyloric development or in contractile function of the pylorus cause reflux of duodenal contents into the stomach and increase the risk of gastric metaplasia and cancer. Abnormalities of the pyloric region are also linked to congenital defects such as the relatively common neonatal hypertrophic pyloric stenosis, and primary duodenogastric reflux. Therefore, understanding pyloric development is of great clinical relevance. Here, we investigated the role of the LIM homeodomain transcription factor Isl1 in pyloric development.
Examination of Isl1 expression in developing mouse stomach by immunohistochemistry, whole mount in situ hybridization and real-time quantitative PCR demonstrated that Isl1 is highly expressed in developing mouse stomach, principally in the smooth muscle layer of the pylorus. Isl1 expression was also examined by immunofluorescence in human hypertrophic pyloric stenosis where the majority of smooth muscle cells were found to express Isl1. Isl1 function in embryonic stomach development was investigated utilizing a tamoxifen-inducible Isl1 knockout mouse model. Isl1 deficiency led to nearly complete absence of the pyloric outer longitudinal muscle layer at embryonic day 18.5, which is consistent with Gata3 null mouse phenotype. Chromatin immunoprecipitation, luciferase assays, and electrophoretic mobility shift assays revealed that Isl1 ensures normal pyloric development by directly targeting Gata3.
This study demonstrates that the Isl1-Gata3 transcription regulatory axis is essential for normal pyloric development. These findings are highly clinically relevant and may help to better understand pathways leading to pyloric disease.
PMCID: PMC4021819  PMID: 24674670
α-smooth muscle actin; Gata3; Isl1; Pylorus
11.  Plumes of neuronal activity propagate in three dimensions through the nuclear avian brain 
BMC Biology  2014;12:16.
In mammals, the slow-oscillations of neuronal membrane potentials (reflected in the electroencephalogram as high-amplitude, slow-waves), which occur during non-rapid eye movement sleep and anesthesia, propagate across the neocortex largely as two-dimensional traveling waves. However, it remains unknown if the traveling nature of slow-waves is unique to the laminar cytoarchitecture and associated computational properties of the neocortex.
We demonstrate that local field potential slow-waves and correlated multiunit activity propagate as complex three-dimensional plumes of neuronal activity through the avian brain, owing to its non-laminar, nuclear neuronal cytoarchitecture.
The traveling nature of slow-waves is not dependent upon the laminar organization of the neocortex, and is unlikely to subserve functions unique to this pattern of neuronal organization. Finally, the three-dimensional geometry of propagating plumes may reflect computational properties not found in mammals that contributed to the evolution of nuclear neuronal organization and complex cognition in birds.
PMCID: PMC4015294  PMID: 24580797
Sleep; Slow waves; Propagation; Travelling; Bird; Cortex
12.  Division in Escherichia coli is triggered by a size-sensing rather than a timing mechanism 
BMC Biology  2014;12:17.
Many organisms coordinate cell growth and division through size control mechanisms: cells must reach a critical size to trigger a cell cycle event. Bacterial division is often assumed to be controlled in this way, but experimental evidence to support this assumption is still lacking. Theoretical arguments show that size control is required to maintain size homeostasis in the case of exponential growth of individual cells. Nevertheless, if the growth law deviates slightly from exponential for very small cells, homeostasis can be maintained with a simple ‘timer’ triggering division. Therefore, deciding whether division control in bacteria relies on a ‘timer’ or ‘sizer’ mechanism requires quantitative comparisons between models and data.
The timer and sizer hypotheses find a natural expression in models based on partial differential equations. Here we test these models with recent data on single-cell growth of Escherichia coli. We demonstrate that a size-independent timer mechanism for division control, though theoretically possible, is quantitatively incompatible with the data and extremely sensitive to slight variations in the growth law. In contrast, a sizer model is robust and fits the data well. In addition, we tested the effect of variability in individual growth rates and noise in septum positioning and found that size control is robust to this phenotypic noise.
Confrontations between cell cycle models and data usually suffer from a lack of high-quality data and suitable statistical estimation techniques. Here we overcome these limitations by using high precision measurements of tens of thousands of single bacterial cells combined with recent statistical inference methods to estimate the division rate within the models. We therefore provide the first precise quantitative assessment of different cell cycle models.
PMCID: PMC4016582  PMID: 24580833
Cell cycle; Bacteria; Division; Size control; Structured population equations; Numerical simulations; Nonparametric estimation
13.  On the extent and role of the small proteome in the parasitic eukaryote Trypanosoma brucei 
BMC Biology  2014;12:14.
Although technical advances in genomics and proteomics research have yielded a better understanding of the coding capacity of a genome, one major challenge remaining is the identification of all expressed proteins, especially those less than 100 amino acids in length. Such information can be particularly relevant to human pathogens, such as Trypanosoma brucei, the causative agent of African trypanosomiasis, since it will provide further insight into the parasite biology and life cycle.
Starting with 993 T. brucei transcripts, previously shown by RNA-Sequencing not to coincide with annotated coding sequences (CDS), homology searches revealed that 173 predicted short open reading frames in these transcripts are conserved across kinetoplastids with 13 also conserved in representative eukaryotes. Mining mass spectrometry data sets revealed 42 transcripts encoding at least one matching peptide. RNAi-induced down-regulation of these 42 transcripts revealed seven to be essential in insect-form trypanosomes with two also required for the bloodstream life cycle stage. To validate the specificity of the RNAi results, each lethal phenotype was rescued by co-expressing an RNAi-resistant construct of each corresponding CDS. These previously non-annotated essential small proteins localized to a variety of cell compartments, including the cell surface, mitochondria, nucleus and cytoplasm, inferring the diverse biological roles they are likely to play in T. brucei. We also provide evidence that one of these small proteins is required for replicating the kinetoplast (mitochondrial) DNA.
Our studies highlight the presence and significance of small proteins in a protist and expose potential new targets to block the survival of trypanosomes in the insect vector and/or the mammalian host.
PMCID: PMC3942054  PMID: 24552149
Genomics; Proteomics; Mass spectrometry data; Non-coding RNA; Mitochondria
14.  Plastid phylogenomics and green plant phylogeny: almost full circle but not quite there 
BMC Biology  2014;12:11.
A study in BMC Evolutionary Biology represents the most comprehensive effort to clarify the phylogeny of green plants using sequences from the plastid genome. This study highlights the strengths and limitations of plastome data for resolving the green plant phylogeny, and points toward an exciting future for plant phylogenetics, during which the vast and largely untapped territory of nuclear genomes will be explored.
PMCID: PMC3925952  PMID: 24533863
15.  Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23 
BMC Biology  2014;12:12.
The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. In this study, we analyzed the role of Awd in controlling Notch signaling during development.
To study the awd gene function we used genetic mosaic approaches to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. Our results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments we show that Notch receptor accumulates in early endosomes in awd mutant follicle cells. A trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation.
In this report we demonstrate that awd is essential for Notch signaling via its endocytic role. In addition, we identify the endocytic step at which Awd function is required for Notch signaling and we obtain evidence indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.
PMCID: PMC3937027  PMID: 24528630
Awd; Notch signaling; Endocytosis
16.  Pax6 regulates the formation of the habenular nuclei by controlling the temporospatial expression of Shh in the diencephalon in vertebrates 
BMC Biology  2014;12:13.
The habenula and the thalamus are two critical nodes in the forebrain circuitry and they connect the midbrain and the cerebral cortex in vertebrates. The habenula is derived from the epithalamus and rests dorsally to the thalamus. Both epithalamus and thalamus arise from a single diencephalon segment called prosomere (p)2. Shh is expressed in the ventral midline of the neural tube and in the mid-diencephalic organizer (MDO) at the zona limitans intrathalamica between thalamus and prethalamus. Acting as a morphogen, Shh plays an important role in regulating cell proliferation and survival in the diencephalon and thalamic patterning. The molecular regulation of the MDO Shh expression and the potential role of Shh in development of the habenula remain largely unclear.
We show that deleting paired-box and homeobox-containing gene Pax6 results in precocious and expanded expression of Shh in the prospective MDO in fish and mice, whereas gain-of-function of pax6 inhibits MDO shh expression in fish. Using gene expression and genetic fate mapping, we have characterized the expression of molecular markers that demarcate the progenitors and precursors of habenular neurons. We show that the thalamic domain is shifted dorsally and the epithalamus is missing in the alar plate of p2 in the Pax6 mutant mouse. Conversely, the epithalamus is expanded ventrally at the expense of the thalamus in mouse embryos with reduced Shh activity. Significantly, attenuating Shh signaling largely rescues the patterning of p2 and restores the epithalamus in Pax6 mouse mutants, suggesting that Shh acts downstream of Pax6 in controlling the formation of the habenula. Similar to that found in the mouse, we show that pax6 controls the formation of the epithalamus mostly via the regulation of MDO shh expression in zebrafish.
Our findings demonstrate that Pax6 has an evolutionarily conserved function in establishing the temporospatial expression of Shh in the MDO in vertebrates. Furthermore, Shh mediates Pax6 function in regulating the partition of the p2 domain into the epithalamus and thalamus.
PMCID: PMC3996077  PMID: 24528677
Organizer activity; Signaling; Forebrain; Habenula; Thalamus; Mice; Zebrafish
17.  Fish populations surviving estrogen pollution 
BMC Biology  2014;12:10.
Among the most common pollutants that enter the environment after passing municipal wastewater treatment are estrogens, especially the synthetic 17α-ethinylestradiol that is used in oral contraceptives. Estrogens are potent endocrine disruptors at concentrations frequently observed in surface waters. However, new genetic analyses suggest that some fish populations can be self-sustaining even in heavily polluted waters. We now need to understand the basis of this tolerance.
See research article:
PMCID: PMC3921986  PMID: 24512617
18.  The cancer-obesity connection: what do we know and what can we do? 
BMC Biology  2014;12:9.
PMCID: PMC3912498  PMID: 24495751
19.  Larval body patterning and apical organs are conserved in animal evolution 
BMC Biology  2014;12:7.
Planktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data.
To compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3β antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the ‘apical plate’. We also unraveled a molecular signature of the apical organ - devoid of six3 but expressing foxj, irx, nkx3 and hox - that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla.
The similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ - comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells - was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae.
PMCID: PMC3939940  PMID: 24476105
Apical-blastoporal axis; Apical organ; Body plan; Larval evolution
20.  Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development 
BMC Biology  2014;12:5.
The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host’s liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood.
Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite’s glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin.
Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.
PMCID: PMC3923246  PMID: 24468049
Cestode; Tapeworm; Echinococcus; Echinococcosis; Insulin; Receptor kinase; Kinase inhibitor; Host-parasite interaction
21.  Uncovering by Atomic Force Microscopy of an original circular structure at the yeast cell surface in response to heat shock 
BMC Biology  2014;12:6.
Atomic Force Microscopy (AFM) is a polyvalent tool that allows biological and mechanical studies of full living microorganisms, and therefore the comprehension of molecular mechanisms at the nanoscale level. By combining AFM with genetical and biochemical methods, we explored the biophysical response of the yeast Saccharomyces cerevisiae to a temperature stress from 30°C to 42°C during 1 h.
We report for the first time the formation of an unprecedented circular structure at the cell surface that takes its origin at a single punctuate source and propagates in a concentric manner to reach a diameter of 2–3 μm at least, thus significantly greater than a bud scar. Concomitantly, the cell wall stiffness determined by the Young’s Modulus of heat stressed cells increased two fold with a concurrent increase of chitin. This heat-induced circular structure was not found either in wsc1Δ or bck1Δ mutants that are defective in the CWI signaling pathway, nor in chs1Δ, chs3Δ and bni1Δ mutant cells, reported to be deficient in the proper budding process. It was also abolished in the presence of latrunculin A, a toxin known to destabilize actin cytoskeleton.
Our results suggest that this singular morphological event occurring at the cell surface is due to a dysfunction in the budding machinery caused by the heat shock and that this phenomenon is under the control of the CWI pathway.
PMCID: PMC3925996  PMID: 24468076
Atomic Force Microscopy (AFM); Saccharomyces cerevisiae; Heat-shock; Cell wall; Chitin; Budding
22.  Flowers and weeds: cell-type specific pruning in the developing visual thalamus 
BMC Biology  2014;12:3.
In the first weeks of vertebrate postnatal life, neural networks in the visual thalamus undergo activity-dependent refinement thought to be important for the development of functional vision. This process involves pruning of synaptic connections between retinal ganglion cells and excitatory thalamic neurons that relay signals on to visual areas of the cortex. A recent report in Neural Development shows that this does not occur in inhibitory neurons, questioning our current understanding of the development of mature neural circuits.
See research article:
PMCID: PMC3903447  PMID: 24468013
23.  Genome-scale reconstruction of the sigma factor network in Escherichia coli: topology and functional states 
BMC Biology  2014;12:4.
At the beginning of the transcription process, the RNA polymerase (RNAP) core enzyme requires a σ-factor to recognize the genomic location at which the process initiates. Although the crucial role of σ-factors has long been appreciated and characterized for many individual promoters, we do not yet have a genome-scale assessment of their function.
Using multiple genome-scale measurements, we elucidated the network of σ-factor and promoter interactions in Escherichia coli. The reconstructed network includes 4,724 σ-factor-specific promoters corresponding to transcription units (TUs), representing an increase of more than 300% over what has been previously reported. The reconstructed network was used to investigate competition between alternative σ-factors (the σ70 and σ38 regulons), confirming the competition model of σ substitution and negative regulation by alternative σ-factors. Comparison with σ-factor binding in Klebsiella pneumoniae showed that transcriptional regulation of conserved genes in closely related species is unexpectedly divergent.
The reconstructed network reveals the regulatory complexity of the promoter architecture in prokaryotic genomes, and opens a path to the direct determination of the systems biology of their transcriptional regulatory networks.
PMCID: PMC3923258  PMID: 24461193
Escherichia coli; Sigma factor; Network reconstruction; Comparative analysis; Klebsiella pneumoniae; Omics data; Systems biology
24.  Populations of a cyprinid fish are self-sustaining despite widespread feminization of males 
BMC Biology  2014;12:1.
Treated effluents from wastewater treatment works can comprise a large proportion of the flow of rivers in the developed world. Exposure to these effluents, or the steroidal estrogens they contain, feminizes wild male fish and can reduce their reproductive fitness. Long-term experimental exposures have resulted in skewed sex ratios, reproductive failures in breeding colonies, and population collapse. This suggests that environmental estrogens could threaten the sustainability of wild fish populations.
Here we tested this hypothesis by examining population genetic structures and effective population sizes (Ne) of wild roach (Rutilus rutilus L.) living in English rivers contaminated with estrogenic effluents. Ne was estimated from DNA microsatellite genotypes using approximate Bayesian computation and sibling assignment methods. We found no significant negative correlation between Ne and the predicted estrogen exposure at 28 sample sites. Furthermore, examination of the population genetic structure of roach in the region showed that some populations have been confined to stretches of river with a high proportion of estrogenic effluent for multiple generations and have survived, apparently without reliance on immigration of fish from less polluted sites.
These results demonstrate that roach populations living in some effluent-contaminated river stretches, where feminization is widespread, are self-sustaining. Although we found no evidence to suggest that exposure to estrogenic effluents is a significant driving factor in determining the size of roach breeding populations, a reduction in Ne of up to 65% is still possible for the most contaminated sites because of the wide confidence intervals associated with the statistical model.
PMCID: PMC3922797  PMID: 24417977
Fishery; Genetic diversity; DNA microsatellites; Waste water treatment work; Ecotoxicology
25.  IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria 
BMC Biology  2013;11:122.
The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown.
Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro.
This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development.
PMCID: PMC3899939  PMID: 24377418
Transcriptional regulator; Corynebacterium glutamicum; Corynebacterium diphtheriae; Mycobacterium tuberculosis; Cell wall synthesis; Mycolic acids; Mycothiol; Inositol; LM; LAM

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