The Epistemic Revolution Induced by Microbiome Studies: An Interdisciplinary View.

Many separate fields and practices nowadays consider microbes as part of their legitimate focus. Therefore, microbiome studies may act as unexpected unifying forces across very different disciplines. Here, we summarize how microbiomes appear as novel major biological players, offer new artistic frontiers, new uses from medicine to laws, and inspire novel ontologies. We identify several convergent emerging themes across ecosystem studies, microbial and evolutionary ecology, arts, medicine, forensic analyses, law and philosophy of science, as well as some outstanding issues raised by microbiome studies across these disciplines and practices. An 'epistemic revolution induced by microbiome studies' seems to be ongoing, characterized by four features: (i) an ecologization of pre-existing concepts within disciplines, (ii) a growing interest in systemic analyses of the investigated or represented phenomena and a greater focus on interactions as their root causes, (iii) the intent to use openly multi-scalar interaction networks as an explanatory framework to investigate phenomena to acknowledge the causal effects of microbiomes, (iv) a reconceptualization of the usual definitions of which individuals are worth considering as an explanans or as an explanandum by a given field, which result in a fifth strong trend, namely (v) a de-anthropocentrification of our perception of the world.

Mapping responsible conduct in the uncharted field of research-creation: A scoping review.

This scoping review addresses the issues of responsible conduct of research (RCR) that can arise in the practice of research-creation (RC), an emergent, interdisciplinary, and heterogeneous field at the interface of academic research and creative activities. Little is yet known about the nature and scope of RCR issues in RC, so our study examined three questions: (1) What are the specific issues in RC in relation to RCR? (2) How does the specificity of RC influence the understanding and practice of RCR? (3) What recommendations could help address the issues highlighted in the literature? To answer these questions, we conducted a scoping review of the academic literature (n = 181 texts) dealing with RCR in RC. We found that researcher-creators faced some very different RCR challenges in comparison with their colleagues in the rest of academia. Addressing these issues is important for both the RCR and RC communities in order to ensure that the rapid development of this field occurs in line with the norms of RCR which, nonetheless, should be adapted to respect the particularities of RC and allow its contributions to the academic world.

Peromyscus populations and their Cuterebra parasites display congruent phylogeographical structure.

The relationships between populations of the Deer Mouse (Peromyscus maniculatus) and the White-footed Mouse (P. leucopus) and their respective Cuterebra parasites were examined. Population genetic structure of hosts and parasites was inferred using cytochrome oxidase mitochondrial sequences of specimens from 7 populations. Genetic analyses revealed that isolation-by-distance applies for P. maniculatus and its associated parasite (C. grisea). A significant correlation was also observed between the genetic distances of these host and parasite species. Furthermore, populations of P. maniculatus and C. grisea from the North and South shores of the St Lawrence River were found to be significantly different. This structure may be explained by the St Lawrence River being a dispersal barrier for both species. A robust analysis of the other species pair (P. leucopus and C. fontinella) could not be performed because of limited sample sizes.

Molecular identification of two species of myiasis-causing Cuterebra by multiplex PCR and RFLP.

The myiasis-causing flies Cuterebra grisea (Coquillet) and Cuterebra fontinella (Clark) (Diptera: Oestridae) are normally parasites of mice, predominantly of the genus Peromyscus. The morphological similarities of these species and the existence of intermediate morphotypes bearing characters of both species make the identification of adults problematic; furthermore the identification of larvae is apparently not possible. This study presents two molecular approaches to discriminate between these species using specific band patterns: (i) species-specific primers designed in the cytochrome oxidase II (COII) region used in multiplex polymerase chain reaction (PCR) and (ii) restriction fragment length polymorphism (RFLP) on amplified segments of cytochrome oxidase I (COI) gene. Both methods were tested on Cuterebra larvae and on adult museum specimens. The two techniques showed a clear difference between C. grisea and C. fontinella, although species-specific primers were more successful than RFLP for degraded DNA. No intraspecific variation in RFLP and species-specific amplifications were detected for the two species of Cuterebra. The results exhibit discrepancies between molecular and morphological identification, suggesting that some of the adults were misidentified.

Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis.

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen's node and rostral primitive streak, at the 6-somite stage ( Charrier, J. B., Teillet, M.-A., Lapointe, F. and Le Douarin, N. M. (1999). Development 126, 4771-4783). In this paper we demonstrate that one day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system.

War and peace in phylogenetics: a rejoinder on total evidence and consensus.

For more than 10 years, systematists have been debating the superiority of character or taxonomic congruence in phylogenetic analysis. In this paper, we demonstrate that the competing approaches can converge to the same solution when a consensus method that accounts for branch lengths is selected. Thus, we propose to use both methods in combination, as a way to corroborate the results of combined and separate analyses. This so-called "global congruence" approach is tested with a wide variety of examples sampled from the literature, and the results are compared with those obtained by standard consensus methods. Our analyses show that when the total evidence and consensus trees differ topologically, collapsing weakly supported nodes with low bootstrap support usually improves "global congruence".

The developmental potentials of the caudalmost part of the neural crest are restricted to melanocytes and glia.

The avian spinal cord is characterized by an absence of motor nerves and sensory nerves and ganglia at its caudalmost part. Since peripheral sensory neurons derive from neural crest cells, three basic mechanisms could account for this feature: (i) the caudalmost neural tube does not generate any neural crest cells; (ii) neural crest cells originating from the caudal part of the neural tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is not permissive for the formation of dorsal root ganglia. To solve this problem, we have first studied the pattern of expression of ventral (HNF3beta) and dorsal (slug) marker genes in the caudal region of the neural tube; in a second approach, we have recorded the emergence of neural crest cells using the HNK1 monoclonal antibody; and finally, we have analyzed the developmental potentials of neural crest cells arising from the caudalmost part of the neural tube in avian embryo in in vitro culture and by means of heterotopic transplantations in vivo. We show here that neural crest cells arising from the neural tube located at the level of somites 47-53 can differentiate both in vitro and in vivo into melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube located caudally to the last pair of somites (i.e. the 53rd pair) does not give rise to neural crest cells in any of the situations tested. The specific anatomical aspect of the avian spinal cord can thus be accounted for by limited developmental potentials of neural crest cells arising from the most caudal part of the neural tube.

Expression of insulin-like growth factor-I (IGF-I) and IGF-II in the avian brain: relationship of in situ hybridization patterns with IGF type 1 receptor expression.

Insulin-like growth factors (IGFs) are expressed in defined spatiotemporal patterns during the development of the mammalian central nervous system (CNS). Since IGF expression in avian species is less well documented, we studied here the expression of IGF-I and IGF-II during chicken CNS development, using in situ hybridization and reverse transcriptase-PCR, and compared the results with the expression of the IGF type 1 receptor (IGF-1R). IGF-II expression started early in embryonic life, shortly after the onset of IGF-1R expression. During organogenesis, IGF-II was strongly expressed in kidney, liver and gut primordia, in contrast with IGF-1R mRNA, which is highly enriched in proliferating neuroepithelia. During the second half of embryonic development, IGF-I and IGF-II had distinct expression patterns, suggesting specific roles for each ligand during brain maturation. IGF-II mRNA was found in numerous brainstem nuclei and in the optic tectum, whereas IGF-I mRNA was found predominantly in telencephalic regions. Both ligands were expressed in the cerebellum, but each by different cell layers. Some brain regions (olfactory bulb and olivo-cerebellar system) did not exhibit the postnatal downregulation typical of extrahepatic IGF-I expression, but continued to express IGF-I into adulthood. Purkinje cells expressed IGF-II in the embryo, but switched to IGF-I expression in the adult. The conservation of embryonic and postnatal IGF expression patterns in the CNS between avians and mammals suggests that the involvement of the IGF system in neurogenesis and differentiation, and possibly in neural plasticity and learning, may have arisen early during tetrapode/vertebrate evolution.

Expression of Frzb-1 during chick development.

We cloned the chick homolog of Xenopus and mouse Frzb-1, a secreted Wnt antagonist and performed in situ hybridizations to determine the pattern of cFrzb-1 expression in the developing chick embryo. At early stages, cFrzb-1 transcripts are located exclusively in the ectodermal layer corresponding to the neural plate. The labelling continues in the neural tube, but is always excluded from the floor plate. cFrzb-1 mRNA is expressed by migrating cephalic and truncal neural crest cells. Later, cFrzb-1 transcripts are found in a subset of neural crest derivatives such as cephalic cartilage, nerves and spinal ganglia. In addition to ectodermal derivatives, cFrzb-1 transcripts were also observed in mesodermal derivatives such as vertebral and limb cartilage, the adrenal cortex, the gonads, and a subpopulation of blood cells.

Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.

Encephalization, adaptation and evolution of chiroptera: A statistical analysis with further evidence for bat monophyly.

As part of a large-scale study on brain morphometrics and adaptations in mammals, we addressed the problem of chiropteran evolution. A specific statistical framework was designed to test which of two competing hypotheses (bat monophyly vs. diphyly) is more strongly supported by quantitative brain data. Our analyses, based on 120 species, revealed that megabats and microbats were more closely related to each other than to primates, and illustrated the convergent adaptations of the brain of bats to similar trophic (i.e. feeding related) niches. Ecologically-corrected characters were then used to derive a new phylogeny which also supports the chiropteran clade. The monophyletic origin of bats is the preferred hypothesis to explain brain quantitative evolution in chiropterans and primates.

Sonic hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud.

Development of the musculature in chick limbs involves tissue and cellular patterning. Patterning at the tissue level leads to the precise arrangement of specific muscles; at the cellular level patterning gives rise to the fibre type diversity in muscles. Although the data suggests that the information controlling muscle patterning is localised within the limb mesenchyme and not in the somitic myogenic precursor cells themselves, the mechanisms underlying muscle organisation have still to be elucidated. The anterior-posterior axis of the limb is specified by a group of cells in the posterior region of the limb mesenchyme, called the zone of polarizing activity (ZPA). When polarizing-region cells are grafted to the anterior margin of the bud, they cause mirror-image digit duplications to be produced. The effect of ZPA grafts can be reproduced by application of retinoic acid (RA) beads and by grafting sonic hedgehog (SHH)-expressing cells to the anterior margin of the limb. Although most previous studies have looked at changes of the skeletal patterning, ZPA and RA also affect muscle patterning. In this report, we investigated the role of SHH in tissue and cellular patterning of forearm wing muscles. Ectopic application of a localised source of SHH to the anterior margin of the wing, leading to complete digit duplication, is able to transform anterior forearm muscles into muscles with a posterior identity. Moreover, the ectopic source of SHH induces a mirror image duplication of the normal posterior muscles fibre types in the new posterior muscles. The reorganisation of the slow fibres can be detected before muscle mass cleavage has started; suggesting that the appropriate fibre type arrangement is in place before the splitting process can be observed.

Total evidence, consensus, and bat phylogeny: A distance-based approach.

Resolution of the total evidence (i.e., character congruence) versus consensus (i.e., taxonomic congruence) debate has been impeded by (1) a failure to employ validation methods consistently across both tree-building and consensus analyses, (2) the incomparability of methods for constructing as opposed to those for combining trees, and (3) indifference to aspects of trees other than their topologies. We demonstrate a uniform, distance-based approach which allows for comparability among the results of character- and taxonomic-congruence studies, whether or not an identical suite of taxa has been included in all contributing data sets. Our results indicate that total-evidence and consensus trees differ little in topology if branch lengths are taken into account when combining two or more trees. In addition, when character-state data are converted to distances, our method permits their combination with information produced by techniques which generate distances directly. Moreover, treating all data sets or trees as distance matrices avoids the problem that different numbers of characters in contributing studies may confound the conclusions of a total-evidence or consensus analysis. Our protocol is illustrated with an example involving bats, in which the three component studies based on serology, DNA hybridization, and anatomy imply distinct phylogenies. However, the total-evidence and consensus trees support a fourth, somewhat different, topology resolved at all but one node and which conforms closely to the currently accepted higher category classification of Chiroptera.

The relationships between notochord and floor plate in vertebrate development revisited.

By using the quail-chicken chimera system, we have previously shown that during development of the spinal cord, floor plate cells are inserted between neural progenitors giving rise to the alar plates. These cells are derived from the regressing Hensen's node or cordoneural hinge (HN-CNH). This common population of HN-CNH cells gives rise to three types of midline descendants: notochord, floor plate, and dorsal endoderm. Here we find that HNF3beta, an important gene in the development of the midline structures, is continuously expressed in the HN-CNH cells and their derivatives, floor plate, notochord, and dorsal endoderm. Experiments in which the notochord was removed in the posterior region of either normal chicken or of quail-chicken chimeras in which a quail HN had been grafted showed that the floor plate develops in a cell-autonomous manner in the absence of notochord. Absence of floor plate observed at the posterior level of the excision results from removal of HN-CNH material, including the future floor plate, and not from the lack of an inductive signal of notochord origin.

Sonic hedgehog is required for survival of both myogenic and chondrogenic somitic lineages.

In vertebrates, the medial moieties of the somites give rise to the vertebrae and epaxial muscles, which develop in close relationship with the axial organs, neural tube and notochord. The lateral moieties contribute to the ribs and to limb and body wall muscles (hypaxial muscles) after a phase of lateral and ventral migration. Surgical ablation of the neural tube and notochord in the chick embryo during segmentation and early differentiation of the somites (day 2 of incubation) does not affect primary development of the hypaxial muscles, but leads to a complete absence of epaxial muscles, vertebrae and ribs, due to cell death in the somites. Here we demonstrate that cell death, which occurs within 24 hours of excision of the axial organs, affects both myogenic and chondrogenic cell lineages defined, respectively, by the expression of MyoD and Pax-1 genes. In contrast, Pax-3 transcripts, normally present in cells giving rise to hypaxial muscles, are preserved in the excised embryos. Backgrafting either the ventral neural tube or the notochord allows survival of MyoD- and Pax-1-expressing cells. Similarly, Sonic hedgehog-producing cells grafted in place of axial organs also rescue MyoD- and Pax-1-expressing cells from death and allow epaxial muscles, ribs and vertebrae to undergo organogenesis. These results demonstrate that the ventral neural tube and the notochord promote the survival of both myogenic and chondrogenic cell lineages in the somites and that this action is mediated by Sonic hedgehog.

The dorsoventral polarity of the presumptive limb is determined by signals produced by the somites and by the lateral somatopleure.

When it first appears at stage HH16, the wing bud is already polarized along the dorsoventral axis. To study the mechanisms leading to the establishment of its dorsoventral polarity, we decided to focus our attention on an earlier stage (HH13). Using the quail-chick chimera system, we first show that the presumptive wing mesoderm occupies the medial half of the somatopleure at the level of somites 15-20. The corresponding ectodermal area, however, will only give rise to the apical ectodermal ridge. The rest of the limb bud ectoderm originates from the ectoderm overlying the paraxial and the intermediate mesoderms for its dorsal aspect and the lateral somatopleural mesoderm for its ventral aspect. We next used five experimental paradigms to show that the dorsoventral polarity of the presumptive limb is determined by its environment. Thus, presumptive limb regions flanked on two sides by rows of somites give rise to bidorsal limb buds, indicating that the somites produce a dorsalizing factor. In addition, insertion of filters laterally to the presumptive limb region also results in bidorsal limb buds, suggesting that the lateral somatopleure produces a ventralizing factor. We propose a model in which the polarizing activity of these two signals is mediated by the morphogenetic movements of the presumptive dorsal and ventral ectoderms, which carry the dorsoventral information over the limb bud mesenchyme.

The avian IGF type 1 receptor: cDNA analysis and in situ hybridization reveal conserved sequence elements and expression patterns relevant for the development of the nervous system.

Insulin-like growth factor type 1 receptor (IGF-1R) is a tyrosine kinase with a key role in development. The primary structure of IGF-1R is known for mammalian species, but not for birds. The avian embryo, however, provides an ideal system for the experimental study of neurogenesis. We therefore cloned the complete coding sequence of the chicken IGF-1R from a cDNA library and analyzed its embryonic expression by Northern blot and in situ hybridization. The deduced chicken IGF-1R precursor of 1363 amino acids was 85% identical to human IGF-1R and did not show deletions or insertions in critical positions, when compared to its mammalian homologues. Notably, all cysteine residues in the extracellular domains, and 15 of the 17 N-linked glycosylation sites found in human IGF-1R were also present in the chicken receptor. An 11 kb transcript was abundant in developing nervous tissues, kidney, pancreas and the gastrointestinal tract. The early in situ expression patterns in 20-somite embryos revealed high levels of IGF-1R mRNA in the neuroepithelia, notochord and somites. At embryonic day 4 (E4), high concentrations of IGF-1R transcripts were found again primarily in the neuroepithelia and, to a lesser degree, in the sensory ganglia and diverse mesenchymal derivatives. During the second half of embryonic development, IGF-1R expression in the CNS was particularly abundant in telencephalic regions, including the olfactory bulb, hippocampus, striatum and piriform cortex, and also in the optic tectum and cerebellum. By the use of cDNA cloning and in situ hybridization this study reveals conserved amino acid sequence elements between birds and mammals, and developmental expression patterns that are compatible with an important role of this receptor in growth, differentiation and maturation of the avian CNS.

Avian olfactory receptors: differentiation of olfactory neurons under normal and experimental conditions.

The bird olfactory system has a simple structure and affords an attractive developmental model for the study of olfactory morphogenesis and differentiation. We have cloned and characterized several chick olfactory receptor (COR) genes belonging to the superfamily of seven-transmembrane domain proteins. In situ hybridization analysis of their spatiotemporal patterns of expression during development reveals several important characteristics. COR expression starts early in placodal cells (Embryonic Day 5, E5). Changes in their expression pattern then correlate with the onset of synaptogenesis (E8). The adult pattern, achieved before hatching, shows that cells expressing a particular COR are not regionalized within the epithelium. By double-label in situ hybridization, we clearly demonstrate that a single cell does not coexpress different COR genes (or subsets of CORs) at any stage of development. Following bulbar deafferentation, COR expression ceases more rapidly than expected from previous axotomy experiments. Concomitantly, a reactivation of the Cash-1 gene, which is involved in early neuronal specification, could be an early sign of olfactory neuronal regeneration. Modulation of COR and Cash-1 expression points to a simultaneous process of neuronal degeneration and regeneration in the olfactory epithelium after axotomy. COR expression is restricted to the olfactory epithelium except during early stages (before synaptogenesis). At that time, cells distributed along the olfactory nerve, from the placode to the anterior telencephalon, also express CORs. This cell population is different from the luteinizing hormone releasing hormone neurons migrating from the placode. Our results show that the olfactory neurons or neuroblasts choose to express one COR before establishing functional connections with the bulb. Later on, bulboepithelial connections seem important not only for olfactory neuron survival but also for stimulation of COR expression. In addition, beyond their implication in functional odor detection, CORs could be involved, at early stages, in processes of olfactory morphogenesis, including the establishment of a bulbar chemotopy.

Rhythmic melatonin secretion in different teleost species: an in vitro study.

The rhythmic production of melatonin is governed by intrapineal oscillators in all fish species so far investigated except the rainbow trout. To determine whether the latter represents an exception among fish, we measured in vitro melatonin secretion in pineal organs of nine wild freshwater and six marine teleost species cultured at constant temperature and under different photic conditions. The results demonstrate that pineal organs of all species maintain a rhythmic secretion of melatonin under light:dark cycles and complete darkness, and strongly suggest that most fish possess endogenous intrapineal oscillators driving the rhythm of melatonin production, with the exception of the rainbow trout.