Evolution of Cyclostome Hox Clusters

Hagfish and lampreys represent the only two lineages of agnathan (jawless) vertebrates that have survived to the present day, and both form the monophyletic group of cyclostomes, which is sister to the rest of the vertebrates, the gnathostomes (jawed vertebrates). Comparisons between cyclostome and gnathostome models are thus essential to infer and understand ancestral traits of vertebrates and their subsequent morphological, genomic, and physiological evolution. It is thought that Hox gene evolution had a major impact in vertebrate morphological diversification, but it has not been until the last decade or so that significant knowledge on the organization and regulation of the Hox clusters of cyclostomes has started to accumulate. In this chapter, we review current data on cyclostome Hox biology, presenting an integrated view of what we know thus far from studies of Hox genes of both the lamprey and hagfish, including their gene complements, cluster evolution, functional and expression diversification, and how this all relates to their body plans and the general vertebrate architecture. We present the similarities and differences with the most well-known gnathostome Hox genes, and infer common, ancestral traits. In conclusion, we propose a common ancestor radically different to previous reconstructions, with only two Hox clusters instead of four, but in which most of the well-known traits of Hox genes –spatial and temporal collinearity, association with cranial segmentation and posterior growth— were already established. This relatively simpler two-cluster ancestor was then independently modified in the cyclostome and gnathostome lineages, where although Hox characteristics have been broadly retained, lineage-specific differences arose over hundreds of millions of years of independent evolution, which could be behind the morphological diversification of each of the groups.Ministerio de Ciencia, Innovación y Universidades, a través del programa Beatriz Galindo a Juan Pascual-Anaya, referencia BEAGAL20/0011

Correlation between vertebral Hox code and vertebral morphology in archosaurs

The evolution of the vertebral column is marked by profound morphological changes that have a strong impact on organismal biology. The vital functions of the axial skeleton range from protecting the neural structures through sustaining the body posture to physiological aspects such as breathing. Archosaurs (crocodiles, birds and dinosaurs), as a group, display a striking variety of body plans and vertebral morphologies. This dissertation aims to contribute to the understanding of the pattern and the genetic basis for the evolution of the vertebral column in archosaurs. The transdisciplinary project comprises five chapters. Framed by a general introduction (chapter 1) and the conclusion (chapter 5), the second chapter considers, from a morphofunctional point of view, the question of (1) why differences in the vertebral column evolved. The present thesis revealed a strong link between the digitally simulated flexion pattern of the presacral vertebral column and the axial movements of modern archosaurs during related activities such as feeding and locomotion: this correlation allowed the inference of the feeding range and locomotor options in the extinct archosaur Plateosaurus. This long-necked dinosaur was primarily adapted as mid-level browser, obtaining food that was at or above the horizontal level of its head. There is currently no evidence to unambiguously interpret the locomotion style of Plateosaurus. The morphofunctional analysis supported both a quadrupedal and a bipedal posture. The third chapter addresses, from a molecular biology point of view, the question (2) of how modern taxa develop their vertebral columns. It provides insights into the genetic basis for the embryonic development of the vertebral column in modern archosaurs, which includes the highly conserved Hox genes. The Hox gene expression pattern was detected in the Nile crocodile (Crocodylus niloticus) via whole-mount in situ hybridisation experiments. Hox paralog genes 4 and 5 are expressed in the cervical region of the crocodile. The anterior expression limit of HoxC-6 marks the cervicothoracic transition. The expression of Hox paralog genes 7 and 8 is restricted to the dorsal series. The same Hox genes are expressed along the anteroposterior body axis of crocodiles, chickens and mice, but the pattern of expression is different. The comparative analysis revealed two general processes that are accompanied by evolutionary differences in the axial skeleton: 1) expansion and condensation as well as 2) a shift of genetic activity corresponding to different vertebral counts. The strong association between the anterior limits of the expression of specific Hox genes and the borders between morphological regions of the vertebral axis in a variety of vertebrate species stimulated the work presented in the fourth chapter. It considers the question (3) of whether we can infer that the development of the vertebral column took place in extinct animals. The direct correlation between vertebral Hox code and quantifiable vertebral morphology shows that the genetic code is deducible from vertebral morphology in modern crocodiles, chickens and mice. Applying these findings to the fossil relative Plateosaurus revealed that the hypothetical Hox code for the dinosaur would be generally similar to the crocodilian Hox gene expression pattern, but with the variation that the anterior region is expanded, as in birds. The integrative analysis (morphology, genes and fossils) of the vertebrae greatly enhanced our knowledge of evolutionary processes and provided valuable information about the possible reasons, genetic basis and pattern for evolutionary changes of the vertebral column in extant and extinct archosaurs.Im Laufe der Evolution hat die Wirbelsäule tiefgreifende morphologische Veränderungen erfahren, die sich signifikant auf die Biologie der Organismen ausgewirkt haben. Die lebenswichtigen Funktionen des Axialskeletts reichen vom Schutz der neuralen Strukturen, über die Stützung des Körpers, bis hin zu physiologischen Aufgaben wie beispielsweise der Atmung. Archosauria (Krokodile, Vögel und Dinosaurier) zeigen eine bemerkenswerte Vielfalt an Körperbauplänen und Wirbelmorphologien. Das Ziel der Dissertation besteht darin, einen entscheidenden Beitrag zum Verständnis der Muster und der genetischen Basis für die Evolution der Wirbelsäule bei Archosauriern zu liefern. Das interdisziplinäre Projekt umfasst fünf Kapitel. Neben einer allgemeinen Einleitung (Kapitel 1) und den Schlussbemerkungen (Kapitel 5), widmet sich das zweite Kapitel aus morphofunktionaler Sicht der Frage (1) warum sich Unterschiede in der Wirbelsäule während der Evolution entwickelt haben. Die vorliegende Arbeit zeigt einen engen Zusammenhang zwischen dem digital simulierten Flexionsmuster der präsakralen Wirbelsäule und den axialen Bewegungen moderner Archosaurier während relevanter Aktivitäten wie beispielsweise Nahrungsaufnahme und Lokomotion. Diese Korrelation ermöglichte es, auf die Nahrungsreichweite sowie die Fortbewegungsmöglichkeiten des ausgestorbenen Archosauriers Plateosaurus rückzuschließen. Dieser langhalsige Dinosaurier war primär als Laubäser auf mittlerem Niveau angepasst, der Nahrung auf oder oberhalb seiner horizontalen Kopfhöhe aufgenommen hat. Es konnte kein eindeutiger Hinweis auf die Fortbewegungsweise von Plateosaurus erbracht werden. Die Ergebnisse der morphofunktionalen Analyse unterstützen sowohl eine quadrupedale als auch eine bipedale Haltung. Das dritte Kapitel behandelt aus molekularbiologischer Sicht die Frage (2) wie moderne Arten ihre Wirbelsäule entwickeln. Es liefert Einsichten in die genetische Basis der embryonalen Entwicklung der Wirbelsäule von modernen Archosauriern; die hoch konservativen Hox Gene. Das Expressionsmuster der Hox Gene wurde beim Nilkrokodil (Crocodylus niloticus) mittels whole-mount in situ Hybridisierungsexperimenten nachgewiesen. Die Hox Gene der paralogen Gruppe 4 und 5 werden in der Halswirbelsäule des Krokodils exprimiert. Die anteriore Expressionsgrenze von HoxC-6 markiert den Übergang von Hals- zu Brustwirbelsäule. Die Hox Gene der paralogen Gruppe 7 und 8 sind auf die Brust- und Lendenwirbelsäule begrenzt. Die gleichen Hox Gene werden entlang der anteroposterioren Körperachse des Krokodils, des Huhns und der Maus exprimiert. Das Muster der Expression ist jedoch unterschiedlich. Die vergleichende Analyse hat zwei generelle Prozesse aufgezeigt, die mit den evolutionären Veränderungen des Axialskeletts in Zusammenhang stehen: 1) die Expansion und Kondensation sowie 2) eine Verschiebung der genetischen Aktivität entsprechend der unterschiedlichen Wirbelanzahl. Der enge Zusammenhang zwischen den anterioren Expressionsgrenzen von spezifischen Hox Genen und den Grenzen zwischen morphologischen Regionen der Wirbelsäule bei einer Vielzahl von Wirbeltierarten regte die Arbeit an, die im vierten Kapitel vorgestellt wird. Es widmet sich der Frage (3) ob man auf die Entwicklung der Wirbelsäule von ausgestorbenen Tieren rückschließen kann. Die direkte Korrelation zwischen dem Hox Code und der quantifizierbaren Wirbelmorphologie zeigt, dass der jeweilige genetische Code von der Wirbelform des modernen Krokodils, Huhns und der Maus ableitbar ist. Diese Ergebnisse wurden auf den fossilen Verwandten Plateosaurus angewendet und lieferten den hypothetischen Hox Code für den Dinosaurier. Er wäre generell ähnlich zum Hox Gen-Expressionsmuster des Krokodils mit der Variation, dass die anteriore Region wie beim Vogel expandiert wäre. Die integrative Analyse (Morphologie, Gene und Fossilien) der Wirbel hat unsere Kenntnis über evolutionäre Prozesse grundlegend erweitert. Sie hat wertvolle Informationen über die möglichen Ursachen, die genetische Basis sowie das Muster der evolutionären Veränderungen der Wirbelsäule heutiger und ausgestorbener Archosaurier geliefert

Anti-Müllerian hormone concentrations in female rabbits and its relation to spay status, pseudopregnancy and ovarian follicle numbers

Anti-Müllerian hormone (AMH), known for its role during foetal sexual differentia- tion, is secreted by the Sertoli cells in males and the granulosa cells in females during post-natal life. As serum AMH concentrations correlate with follicle numbers, AMH is utilized as a marker of ovarian reserve in many species. In dogs and cats, AMH is used as a diagnostic tool to determine spay or neuter status. In the available literature, no research regarding serum AMH levels in rabbits has been published yet. The objec- tives of the present study were to (1) measure serum AMH concentrations in female rabbits and investigate the value of AMH as a diagnostic tool to differentiate between spayed and intact does and (2) relate measured AMH levels to pseudopregnancy and ovarian follicle numbers. For AMH measurement, serum samples were obtained from sexually intact (n = 64) and spayed ( n = 22) female rabbits. Spayed does were of various breeds; intact rabbits were Zika hybrid rabbits. In the intact does, AMH measurement was complemented by determination of progesterone levels, gynae - cological examination and histopathological evaluation of the uterus and ovaries, in- cluding follicle counts. Serum AMH and progesterone concentrations were measured using a human-based chemiluminescence immunoassay (CLIA) and an enzyme-linked fluorescence assay (ELFA), respectively. Depending on progesterone levels, sexually intact does were classified into follicular (n = 52) or luteal phase ( n = 12). Median serum AMH levels were 1.53 ng/ml (range 0.77–3.36 ng/ml) in intact and 0.06 ng/ ml (range ≤0.01–0.23 ng/ml) in spayed does. AMH concentrations between the in- tact and spayed rabbits differed significantly and did not overlap (p < .001). Receiver operating characteristic (ROC) curve analysis yielded a sensitivity and specificity of 100% for a cut-off level of 0.50 ng/ml. Follicular or luteal phase had no significant influence on measured AMH levels (t = 0.061, df = 62, p = .951). While the number of secondary follicles correlated significantly with AMH concentrations (r s = 0.410, p = .001), the number of primary or antral follicles did not ( r s = 0.241, p = .055 and r s = 0.137, p = .281, respectively). In conclusion, a single determination of serum AMH concentrations was adequate to distinguish spayed from intact female rabbits. Among sexually intact individuals, whether does were in follicular or luteal phase had no sig - nificant influence on measured serum AMH concentrations. The relationship between small growing follicles and AMH levels as described in other species could be partially confirmed, as secondary follicles correlated significantly with AM

Modelling, design and control of a bird neck using tensegrity mechanisms

International audienceIn birds, the neck exhibits remarkable performances and serves as a dextrous arm for performing various tasks. Accordingly, it is an interesting bioinspiration for designing new manipulators with enhanced performances. This paper proposes a preliminary bird neck model using several stacked tensegrity crossed bar mechanisms. It addresses several issues regarding kinetostatic and dynamic modelling, design and control

Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus

BACKGROUND: The GP5+/GP6+ PCR assay is a well-established HPV detection technique. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. METHODS: Firstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng–100 ng) diluted in a background of C-33A DNA (100 ng-2 μg). Secondly, the detection of small quantities (15ag-1.5pg) of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56) diluted in C-33A DNA was investigated. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors) were tested for HPV. Six different PCR protocols were assessed. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. RESULTS: HPV detection sensitivity was dependent on the total amount of DNA in a PCR. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 μg or 1 μg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (~28 copies HPV-16) in 500 ng or 100 ng background DNA. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 μg or 1 μg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. HPV recombinant plasmids were each detected with high (albeit varying) sensitivity by a touchdown protocol. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA) than by a touchdown approach (15fg detection limit). HPV-52 was not amplified by the standard protocol at the dilutions tested. Seventeen different HPV types were demonstrated in 47/65 (72%) abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. Twenty-one different HPV types were recorded in 111/114 (97%) vaginal lesions. Multiple infections were also detectable using a touchdown approach. Of 26 breast tumors, 5 (19%) tested HPV positive by the standard assay and 15/26 (58%) using a touchdown protocol. CONCLUSION: Touchdown modification of the GP5+/GP6+ PCR assay enables the detection of HPV undetected under regular assay conditions. The use of standardized DNA quantities in a PCR rather than standard sample volumes containing arbitrary amounts of DNA is supported. A touchdown approach may be beneficial as an analytical test for the re-evaluation of (apparently) HPV negative abnormal cervical cytological or histological samples, and for investigating the association of HPV with disease conditions at diverse organ sites. The clinical utility of a touchdown approach for HPV detection requires further investigation as increased assay analytical sensitivity may not necessarily equate with improved clinical sensitivity or specificity

Homeotic transformations reflect departure from the mammalian 'rule of seven' cervical vertebrae in sloths: inferences on the Hox code and morphological modularity of the mammalian neck

Background: Sloths are one of only two exceptions to the mammalian 'rule of seven' vertebrae in the neck. As a striking case of breaking the evolutionary constraint, the explanation for the exceptional number of cervical vertebrae in sloths is still under debate. Two diverging hypotheses, both ultimately linked to the low metabolic rate of sloths, have been proposed: hypothesis 1 involves morphological transformation of vertebrae due to changes in the Hox gene expression pattern and hypothesis 2 assumes that the Hox gene expression pattern is not altered and the identity of the vertebrae is not changed. Direct evidence supporting either hypothesis would involve knowledge of the vertebral Hox code in sloths, but the realization of such studies is extremely limited. Here, on the basis of the previously established correlation between anterior Hox gene expression and the quantifiable vertebral shape, we present the morphological regionalization of the neck in three different species of sloths with aberrant cervical count providing indirect insight into the vertebral Hox code. Results: Shape differences within the cervical vertebral column suggest a mouse-like Hox code in the neck of sloths. We infer an anterior shift of HoxC-6 expression in association with the first thoracic vertebra in short-necked sloths with decreased cervical count, and a posterior shift of HoxC-5 and HoxC-6 expression in long-necked sloths with increased cervical count. Conclusion: Although only future developmental analyses in non-model organisms, such as sloths, will yield direct evidence for the evolutionary mechanism responsible for the aberrant number of cervical vertebrae, our observations lend support to hypothesis 1 indicating that the number of modules is retained but their boundaries are displaced. Our approach based on quantified morphological differences also provides a reliable basis for further research including fossil taxa such as extinct 'ground sloths' in order to trace the pattern and the underlying genetic mechanisms in the evolution of the vertebral column in mammals

Association of genomic variants at the human leukocyte antigen locus with cervical cancer risk, HPV status and gene expression levels

The human leukocyte antigen (HLA) locus on chromosome 6 has been reported to be associated with cervical cancer. We investigated two independent single‐nucleotide polymorphisms in a large case‐control series of cervical dysplasia and carcinoma that has been newly established by the German Cervigen Consortium, comprising a total of 2481 cases and 1556 healthy females. We find significant associations for both variants, rs9272117 at HLA‐DQA1 and rs2844511 at MICA and HCP5, with cervical disease. Both variants showed evidence of association with invasive cervical cancer (rs9272117: OR 0.89, 95% CI 0.79‐0.99, P = .036; rs2844511: OR 1.17, 95% CI 1.04‐1.31, P = .008) and with high‐grade dysplasia (rs9272117: OR 0.78, 95% CI 0.70‐0.87, P = 7.1 × 10−6; rs2844511: OR 1.13, 95% CI 1.01‐1.26, P = .035), as well as in a combined analysis of both groups (rs9272117: OR 0.83, 95% CI 0.75‐0.91, P = 6.9 × 10−5; rs2844511: OR 1.14, 95% CI 1.04‐1.26, P = .005). Variant rs2844511, but not rs9272117, also showed modest evidence of association with low‐grade dysplasia (OR 1.26, 95% CI 1.04‐1.54, P = .019). In case‐only analyses, rs2844511 tended to predict HPV status (P = .044) and rs9272117 tended to associate with HPV16 (P = .022). RNA studies in cervical samples showed a significant correlation in the transcript levels of MICA, HCP5 and HLA‐DQA1, suggesting extensive co‐regulation. All three genes were upregulated in HPV16‐positive samples. In stratified analyses, rs9272117 was associated with HLA‐DQA1 levels, specifically in HPV‐positive samples, while rs2844511 was associated with MICA and HCP5 levels. The risk allele of rs2844511 was required for correlations between MICA or HCP5 with HLA‐DQA1. Altogether, our results support 6p21.32‐33 as the first consistent cervical cancer susceptibility locus and provide evidence for a link between genetic risk variants, HPV16 status and transcript levels of HLA‐DQA1, HCP5 and MICA, which may contribute to tumor immune evasion

New horizons for fundamental physics with LISA

The Laser Interferometer Space Antenna (LISA) has the potential to reveal wonders about the fundamental theory of nature at play in the extreme gravity regime, where the gravitational interaction is both strong and dynamical. In this white paper, the Fundamental Physics Working Group of the LISA Consortium summarizes the current topics in fundamental physics where LISA observations of gravitational waves can be expected to provide key input. We provide the briefest of reviews to then delineate avenues for future research directions and to discuss connections between this working group, other working groups and the consortium work package teams. These connections must be developed for LISA to live up to its science potential in these areas

Skull Shape Diversity in Pet Rabbits and the Applicability of Anatomical Reference Lines for Objective Interpretation of Dental Disease

Acquired dental problems are among the most frequently encountered diseases in pet rabbits. However, early symptoms are often overlooked because the affected animals first appear completely asymptomatic. Alterations from anatomical reference lines according to B&ouml;hmer and Crossley applied to standard skull X-ray images, have been shown to be indicative of tooth health problems in pet rabbits. Despite its proven usefulness, there are exceptions in which the anatomical reference lines appear not to be suitable for application. We addressed this issue by quantifying the cranial morphology of a large data set of pet rabbit patients (N = 80). The results of the morphometric analyses revealed considerable diversity in skull shape among the typical pet rabbits, but variance in only a few parameters influences the applicability of the anatomical reference lines. The most substantial parameter is the palatal angle. Specimens in which the anatomical reference lines could not be applied, have a rather large angle between the skull base and the palatal bone. We recommend to measure the palatal angle before applying the anatomical reference lines for objective interpretation of dental disease. Pet rabbits with a palatal angle larger than 18.8&deg; are not strictly suitable for the successful application of the anatomical reference lines

Correlation between Hox code and vertebral morphology in the mouse: towards a universal model for Synapsida

Abstract Background The importance of the cervical vertebrae as part of the skull–neck system in facilitating the success and diversity of tetrapods is clear. The reconstruction of its evolution, however, is problematic because of the variation in the number of vertebrae, making it difficult to identify homologous elements. Quantification of the morphological differentiation in the neck of diverse archosaurs established homologous units of vertebrae (i.e. modules) resulting from Hox gene expression patterns within the cervical vertebral column. The present study aims to investigate the modularity of the cervical vertebral column in the mouse and to reveal the genetic patterns and changes underlying the evolution of the neck of modern mammals and their extinct relatives. In contrast to modern mammals, non-mammalian synapsids are characterized by a variable cervical count, the presence of free cervical ribs and the presence of a separate CV1 centrum. How might these evolutionary modifications be associated with changes in the Hox code? Results In combination with up-to-date information on cervical Hox gene expression including description of the vertebral phenotype of Hox knock-out mutants, the 3D landmark-based geometric morphometric approach demonstrates a correlation between Hox code and vertebral morphology in the mouse. There is evidence that the modularity of the neck of the mouse had already been established in the last common ancestor of mammals, but differed from that of non-mammalian synapsids. The differences that likely occurred during the evolution of synapsids include an anterior shift in HoxA-5 expression in relation to the reduction of cervical ribs and an anterior shift in HoxD-4 expression linked to the development of the highly differentiated atlas-axis complex, whereas the remaining Hox genes may have displayed a pattern similar to that in mammals on the basis of the high level of conservatism in the axial skeleton of this lineage. Conclusion Thus, the mouse Hox code provides a model for understanding the evolutionary mechanisms responsible for the great morphological adaptability of the cervical vertebral column in Synapsida. However, more studies in non-model organisms are required to further elucidate the evolutionary role of Hox genes in axial patterning of the unique mammalian body plan