Glia-Mediated Regenerative Response Following Acute Excitotoxic Damage in the Postnatal Squamate Retina

The retina is a complex tissue responsible for both detection and primary processing of visual stimuli. Although all vertebrate retinas share a similar, multi-layered organization, the ability to regenerate individual retinal cells varies tremendously, being extremely limited in mammals and birds when compared to anamniotes such as fish and amphibians. However, little is yet known about damage response and regeneration of retinal tissues in "non-classical" squamate reptiles (lizards, snakes), which occupy a key phylogenetic position within amniotes and exhibit unique regenerative features in many tissues. Here, we address this gap by establishing and characterizing a model of excitotoxic retinal damage in bearded dragon lizard (Pogona vitticeps). We particularly focus on identifying, at the cellular and molecular level, a putative endogenous cellular source for retinal regeneration, as diverse self-repair strategies have been characterized in vertebrates using a variety of retinal injury and transgenic models. Our findings reveal for the first time that squamates hold the potential for postnatal retinal regeneration following acute injury. Although no changes occur in the activity of physiologically active progenitors recently identified at the peripheral retinal margin of bearded dragon, two distinct successive populations of proliferating cells at central retina respond to neurotoxin treatment. Following an initial microglia response, a second source of proliferating cells exhibit common hallmarks of vertebrate Muller glia (MG) activation, including cell cycle re-entry, dedifferentiation into a progenitor-like phenotype, and re-expression of proneural markers. The observed lizard glial responses, although not as substantial as in anamniotes, appear more robust than the absent or neonatal-limited regeneration reported without exogenous stimulation in other amniotes. Altogether, these results help to complete our evolutionary understanding of regenerative potential of the vertebrate retina, and further highlight the major importance of glial cells in retinal regeneration. Furthermore, our work offers a new powerful vertebrate model to elucidate the developmental and evolutionary bases of retinal regeneration within amniotes. Such new understanding of self-repair mechanisms in non-classical species endowed with regenerative properties may help designing therapeutic strategies for vertebrate retinal diseases.Peer reviewe

Microzooplankton grazing impact along a trophic gradient from the Atlantic Ocean to the Western Mediterranean Sea

2008/2009Nell'ambito della crociera transmediterranea facente parte del progetto V.E.C.T.O.R. è stato quantificato il flusso di carbonio che veicola attraverso le reti trofiche pelagiche tramite la predazione del microzooplancton su un ampio spettro di prede; si è inoltre verificato la selezione delle prede e quantificato la produzione secondaria. Per questa analisi è stato adottato il metodo delle diluizioni introdotto da Landry ed Hassett (1982)che ci permette di stimare sia il tasso di crescita delle prede, che quello di mortalità delle prede indotta da predazione. In concomitanza con gli esperimenti di diluizione,si è valutato la comunità dei predatori con differenti metodi di campionamento e di conservazione dei campioni nonchè, l'attività di predazione del mesozooplancton sul comparto microplanctonico nel Mar Ligure e nel Mar Tirreno. I risultati complessivi della ricerca indicano che in condizioni di estrema oligotrofia nelle quali è avvenuta la crociera nell'estate 2007, il flusso di carbonio di maggiore entità che fluisce attraverso il comparto microzooplanctonico deriva dalle biomasse dei batteri eterotrofi, che sono le uniche consistenti in tutta l'area. Nanoplancton e microfitoplancton, contribuiscono scarsamente alla dieta del microzooplancton. Dallo studio quali-quantitativo del microooplancton si evince che la rappresentazione migliore in termini di ricchezza si ottiene con maggiori volumi di campionamento (5 L, conservati in formalina al 2%) mentre, l'abbondanza più rilevante si riscontra quando i campioni (300 mL) vengono conservati in soluzione di Lugol al 2%. I campioni ottenuti dall'esperimento di diluizione (2 L, conservati in formalina al 2%) offrono nel complesso la miglior rappresentazione della comunità dei predatori. Nel Mar Ligure, il calanoide Centropages esercita il suo impatto di predazione sia sul comparto microzooplanctonico che su quello microfitoplanctonico, sebbene sia indirizzato in questo caso, unicamente alla categoria dei dinoflagellati a dimensioni maggiori e ai coccolitoforidi. Nel Mar Tirreno, il ciclopoide Corycaeus esercita la sua selezione unicamente sul comparto microzooplanctonico. Per la stazione tirrenica si registrano valori di export seppur minimi. Nel complesso, sembra che in condizioni di oligotrofia, l'energia trasferita ai livelli trofici superiori sia molto esigua.In the frame of the V.E.C.T.O.R. project, during the transmediterranean cruise, the carbon fluxes throughout the pelagic trophic webs were quantified by means of the grazing impact of microzooplankton on a wide variety of prey; The selection exerted by microzooplankton on its prey and the secondary production was also estimated. The dilution method introduced by Landry and hassett (1982) was applied to the research. The method assess both the growth of the prey and their mortality induced by grazing. Beside dilution experiments, a quali-quantitative analysis of the predators comunity treated with different fixatives and sampling techniques was performed. In the Ligurian and in the Tyrrhenian Sea, a further examination on mesozooplankton grazing on microplankton assemblages was estimated. Risults confirm that in extreme oligotrophic conditions as those occured during summer 2007, the major carbon flux fuelled through microzooplankton community derived from heterotrophic bacteria, that showed the most abundant biomasses along the sampled stations. Nano- and microphytoplankton were of minor importance in the diet of their direct grazers. From the quali-quantitative analysis of microzooplankton, the best representation in terms of species richness is obtained by means of higher sampling volumes (5 L, fixed in 2% formalin) whereas, the higher abundances were displayed for samples collected in 300 mL and conserved in Lugol's solution. Samples derived from dilution experiments (2 L fixed in 2% formalin) among all, displayed the best representation of the predators community. In the Ligurian Sea, the calanoid Centropages, exerted its grazing impact on both microzooplankton and microphytoplankton; on the latters, the selection is addressed only on dinoflagellates of bigger size and on coccolithophorids. In the Tyrrhenian Sea, the cyclopoid Corycaeus, selects all microzooplankton assemblage but aloricate ciliates. The Tyrrhenian Sea, recorded a minimum export. In oligotrophic conditions, the energy fuelled to the higher trophic levels seems to be very scarce.XXII Ciclo197

On varieties with higher osculating defect

In this paper, using the method of moving frames, we generalise some of Terracini's results on varieties with tangent defect. In particular, we characterise varieties with higher order osculating defect in terms of Jacobians of higher fundamental forms and moreover we characterise varieties with "small" higher fundamental forms as contained in scrolls.Comment: AMS-LaTeX; 17 pages. To appear in Revista Matem\'atica Iberoam\'erican

Embryonic development of skull bones in the Sahara horned viper (Cerastes cerastes), with new insights into structures related to the basicranium and braincase roof

Ontogenetic studies are crucial for understanding functional morphology, origin and adaptation of skulls in vertebrates. However, very few studies have so far released complete embryonic series focusing on skull embryonic development in species showing diverse and extreme cranial morphologies such as snakes. The wide distribution and unique reproductive and ecological behaviors of venomous vipers, including the heterogeneity in breeding and egg incubation periods in oviparous species, make this group an excellent new model for studying the diversity of skull developmental processes in snakes. Here we present the first complete description of osteocranium development in a viperine snake, Cerastes cerastes, using detailed analysis of the ossification pattern of individual bones across different embryonic stages based on high-resolution micro-computed tomography data. Particularly, we describe in detail the development of the laterosphenoid from its dorsal and ventral components, dividing the trigeminal foramen into maxillary and mandibular foramina. Furthermore, our data help clarify some controversy concerning the presence and/or origin of structures related to the snake basicranium and braincase roof. For example, our detailed description of supraoccipital development suggests that this bone derived, at least in part, from the tectum posterius, although the involvement of the tectum synoticum cannot be totally excluded. Similarly, the epiotic centers of supraoccipital ossification are confirmed during braincase development, and the ancestral lacrimal bone primordium is observed as a ventral element at the early stages of prefrontal development. Finally, our embryonic C. cerastes data highlight a plausible asymmetry in snake skull development, mostly occurring in the basicranium region, but further investigations of embryonic samples and viper species would be required to confirm such phenomenon.Peer reviewe

Comparative analysis of squamate brains unveils multi-level variation in cerebellar architecture associated with locomotor specialization

Ecomorphological studies evaluating the impact of environmental and biological factors on the brain have so far focused on morphology or size measurements, and the ecological relevance of potential multi-level variations in brain architecture remains unclear in vertebrates. Here, we exploit the extraordinary ecomorphological diversity of squamates to assess brain phenotypic diversification with respect to locomotor specialization, by integrating single-cell distribution and transcriptomic data along with geometric morphometric, phylogenetic, and volumetric analysis of high-definition 3D models. We reveal significant changes in cerebellar shape and size as well as alternative spatial layouts of cortical neurons and dynamic gene expression that all correlate with locomotor behaviours. These findings show that locomotor mode is a strong predictor of cerebellar structure and pattern, suggesting that major behavioural transitions in squamates are evolutionarily correlated with mosaic brain changes. Furthermore, our study amplifies the concept of 'cerebrotype', initially proposed for vertebrate brain proportions, towards additional shape characters.Peer reviewe

The alternative regenerative strategy of bearded dragon unveils the key processes underlying vertebrate tooth renewal

Deep understanding of tooth regeneration is hampered by the lack of lifelong replacing oral dentition in most conventional models. Here, we show that the bearded dragon, one of the rare vertebrate species with both polyphyodont and monophyodont teeth, constitutes a key model for filling this gap, allowing direct comparison of extreme dentition types. Our developmental and high-throughput transcriptomic data of microdissected dental cells unveils the critical importance of successional dental lamina patterning, in addition to maintenance, for vertebrate tooth renewal. This patterning process happens at various levels, including directional growth but also gene expression levels, dynamics, and regionalization, and involves a large number of yet uncharacterized dental genes. Furthermore, the alternative renewal mechanism of bearded dragon dentition, with dual location of slow-cycling cells, demonstrates the importance of cell migration and functional specialization of putative epithelial stem/progenitor niches in tissue regeneration, while expanding the diversity of dental replacement strategies in vertebrates.Peer reviewe

Ontogeny of Hemidactylus (Gekkota, Squamata) with emphasis on the limbs

Squamate reptiles constitute a major component of the world's terrestrial vertebrate diversity, encompassing many morphotypes related to ecological specialization. Specifically, Gekkota, the sister clade to most other squamates, have highly specialized autopodia, which have been linked to their ecological plasticity. In this study, a developmental staging table of the gecko Hemidactylus, housed at the Museum fur Naturkunde, is established. Twelve post-ovipositional stages are erected, monitoring morphological embryological transitions in eye, ear, nose, heart, limbs, pharyngeal arches, and skin structures. Ecomorphological specializations in the limbs include multiple paraphalanges, hypothesized to aid in supporting the strong muscles, that are situated adjacent to metacarpal and phalangeal heads. Furthermore, some phalanges are highly reduced in manual digits III and IV and pedal digits III, IV, and V. Development, composition, and growth of limb elements is characterized in detail via mu CT, histochemistry, and bone histological analysis. Using known life history data from two individuals, we found an average lamellar bone accretion rate in the humeral diaphysis comparable to that of varanids. Various adult individuals also showed moderate to extensive remodeling features in their long bone cortices, indicating that these animals experience a highly dynamic bone homeostasis during their growth, similar to some other medium-sized to large squamates. This study of in-ovo development of the gecko Hemidactylus and its ecomorphological specializations in the adult autopodia, enlarges our knowledge of morphological trait evolution and of limb diversity within the vertebrate phylum.Peer reviewe

The anatomical placode in reptile scale morphogenesis indicates shared ancestry among skin appendages in amniotes

Most mammals, birds, and reptiles are readily recognized by their hairs, feathers, and scales, respectively. However, the lack of fossil intermediate forms between scales and hairs and substantial differences in their morphogenesis and protein composition have fueled the controversy pertaining to their potential common ancestry for decades. Central to this debate is the apparent lack of an “anatomical placode” (that is, a local epidermal thickening characteristic of feathers’ and hairs’ early morphogenesis) in reptile scale development. Hence, scenarios have been proposed for the independent development of the anatomical placode in birds and mammals and parallel co-option of similar signaling pathways for their morphogenesis. Using histological and molecular techniques on developmental series of crocodiles and snakes, as well as of unique wild-type and EDA (ectodysplasin A)–deficient scaleless mutant lizards, we show for the first time that reptiles, including crocodiles and squamates, develop all the characteristics of an anatomical placode: columnar cells with reduced proliferation rate, as well as canonical spatial expression of placode and underlying dermal molecular markers. These results reveal a new evolutionary scenario where hairs, feathers, and scales of extant species are homologous structures inherited, with modification, from their shared reptilian ancestor’s skin appendages already characterized by an anatomical placode and associated signaling molecules.Peer reviewe

The developmental origins of heterodonty and acrodonty as revealed by reptile dentitions

Despite the exceptional diversity and central role of dentitions in vertebrate evolution, many aspects of tooth characters remain unknown. Here, we exploit the large array of dental phenotypes in acrodontan lizards, including EDA mutants showing the first vertebrate example of positional transformation in tooth identity, to assess the developmental origins and evolutionary patterning of tooth types and heterodonty. We reveal that pleurodont versus acrodont dentition can be determined by a simple mechanism, where modulation of tooth size through EDA signaling has major consequences on dental formula, thereby providing a new flexible tooth patterning model. Furthermore, such implication of morphoregulation in tooth evolution allows predicting the dental patterns characterizing extant and fossil lepidosaurian taxa at large scale. Together, the origins and diversification of tooth types, long a focus of multiple research fields, can now be approached through evo-devo approaches, highlighting the importance of underexplored dental features for illuminating major evolutionary patterns.Peer reviewe

Skull Development, Ossification Pattern, and Adult Shape in the Emerging Lizard Model Organism Pogona vitticeps: A Comparative Analysis With Other Squamates

The rise of the Evo-Devo field and the development of multidisciplinary research tools at various levels of biological organization have led to a growing interest in researching for new non-model organisms. Squamates (lizards and snakes) are particularly important for understanding fundamental questions about the evolution of vertebrates because of their high diversity and evolutionary innovations and adaptations that portrait a striking body plan change that reached its extreme in snakes. Yet, little is known about the intricate connection between phenotype and genotype in squamates, partly due to limited developmental knowledge and incomplete characterization of embryonic development. Surprisingly, squamate models have received limited attention in comparative developmental studies, and only a few species examined so far can be considered as representative and appropriate model organism for mechanistic Evo-Devo studies. Fortunately, the agamid lizard Pogona vitticeps (central bearded dragon) is one of the most popular, domesticated reptile species with both a well-established history in captivity and key advantages for research, thus forming an ideal laboratory model system and justifying his recent use in reptile biology research. We first report here the complete post-oviposition embryonic development for P. vitticeps based on standardized staging systems and external morphological characters previously defined for squamates. Whereas the overall morphological development follows the general trends observed in other squamates, our comparisons indicate major differences in the developmental sequence of several tissues, including early craniofacial characters. Detailed analysis of both embryonic skull development and adult skull shape, using a comparative approach integrating CT-scans and gene expression studies in P. vitticeps as well as comparative embryology and 3D geometric morphometrics in a large dataset of lizards and snakes, highlights the extreme adult skull shape of P. vitticeps and further indicates that heterochrony has played a key role in the early development and ossification of squamate skull bones. Such detailed studies of embryonic character development, craniofacial patterning, and bone formation are essential for the establishment of well-selected squamate species as Evo-Devo model organisms. We expect that P. vitticeps will continue to emerge as a new attractive model organism for understanding developmental and molecular processes underlying tissue formation, morphology, and evolution.Peer reviewe