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Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Instituto Gulbenkian de Ciência

Faculdade de Ciências da Universidade de Lisboa

Dicynodontia represent the most diverse tetrapod group during the Late Permian. They survived the Permo-Triassic extinction and are central to understanding Permo-Triassic terrestrial ecosystems. Although extensively studied, several aspects of dicynodont paleobiology such as, neuroanatomy, inner ear morphology and internal cranial anatomy remain obscure. Here we describe a new dicynodont (Therapsida, Anomodontia) from northern Mozambique: Niassodon mfumukasi gen. et sp. nov. The holotype ML1620 was collected from the Late Permian K5 formation, Metangula Graben, Niassa Province northern Mozambique, an almost completely unexplored basin and country for vertebrate paleontology. Synchrotron radiation based micro-computed tomography (SRµCT), combined with a phylogenetic analysis, demonstrates a set of characters shared with Emydopoidea. All individual bones were digitally segmented allowing a 3D visualization of each element. In addition, we reconstructed the osseous labyrinth, endocast, cranial nerves and vasculature. The brain is narrow and the cerebellum is broader than the forebrain, resembling the conservative, "reptilian-grade" morphology of other non-mammalian therapsids, but the enlarged paraflocculi occupy the same relative volume as in birds. The orientation of the horizontal semicircular canals indicates a slightly more dorsally tilted head posture than previously assumed in other dicynodonts. In addition, synchrotron data shows a secondary center of ossification in the femur. Thus ML1620 represents, to our knowledge, the oldest fossil evidence of a secondary center of ossification, pushing back the evolutionary origins of this feature. The fact that the specimen represents a new species indicates that the Late Permian tetrapod fauna of east Africa is still incompletely known. 

Bringing dicynodonts back to life: paleobiology and anatomy of a new emydopoid genus from the Upper Permian of Mozambique.

OpenSPIM and OpenSpinMicroscopy emerged as open access platforms for Light Sheet and Optical Projection Imaging, often called as optical mesoscopy techniques. Both projects can be easily reproduced using comprehensive online instructions that should foster the implementation and further development of optical imaging techniques with sample rotation control. This additional dimension in an open system offers the possibility to make multi-view microscopy easily modified and will complement the emerging commercial solutions. Furthermore, it is deeply based on other open platforms such as MicroManager and Arduino, enabling development of tailored setups for very specific biological questions. In our perspective, the open access principle of OpenSPIM and OpenSpinMicroscopy is a game-changer, helping the concepts of light sheet and optical projection tomography (OPT) to enter the mainstream of biological imaging.

Going "open" with mesoscopy: a new dimension on multi-view imaging.

The Japanese quail is a widely used model organism for the study of embryonic development; however, anatomical resources are lacking. The Quail Anatomy Portal (QAP) provides 22 detailed three-dimensional (3D) models of quail embryos during development from embryonic day (E)1 to E15 generated using optical projection tomography. The 3D models provided can be virtually sectioned to investigate anatomy. Furthermore, using the 3D nature of the models, we have generated a tool to assist in the staging of quail samples. Volume renderings of each stage are provided and can be rotated to allow visualization from multiple angles allowing easy comparison of features both between stages in the database and between images or samples in the laboratory. The use of JavaScript, PHP and HTML ensure the database is accessible to users across different operating systems, including mobile devices, facilitating its use in the laboratory.The QAP provides a unique resource for researchers using the quail model. The ability to virtually section anatomical models throughout development provides the opportunity for researchers to virtually dissect the quail and also provides a valuable tool for the education of students and researchers new to the field. Database URL: http://quail.anatomyportal.org (For review username: demo, password: quail123).

The quail anatomy portal.

The stereotypic left-right (LR) asymmetric distribution of internal organs is due to an asymmetric molecular cascade in the lateral plate mesoderm (LPM) that is originated at the embryonic node. In chicken embryos, molecular asymmetries at Hensen's node are created by leftward cell movements that occur transiently. What terminates these movements, and, moreover, what is the impact of prolonging them on the LR asymmetry cascade? We show that leftward movements last longer when N-cadherin function is blocked and cease prematurely when N-cadherin is overexpressed on the right side of the node. The prolonged leftward movements lead to loss of asymmetric expression of fgf8 and nodal at the node region. This originates an abnormal expression of the asymmetric genes cer1 and snai1 in the LPM, resulting in mispositioned hearts. We conclude that N-cadherin stops the leftward cell movements and that this termination is an essential step in the establishment of LR asymmetry. 

N-cadherin locks left-right asymmetry by ending the leftward movement of Hensen's node cells.

Leptin is a hormone produced by the adipose tissue that acts in the brain, stimulating white fat breakdown. We find that the lipolytic effect of leptin is mediated through the action of sympathetic nerve fibers that innervate the adipose tissue. Using intravital two-photon microscopy, we observe that sympathetic nerve fibers establish neuro-adipose junctions, directly "enveloping" adipocytes. Local optogenetic stimulation of sympathetic inputs induces a local lipolytic response and depletion of white adipose mass. Conversely, genetic ablation of sympathetic inputs onto fat pads blocks leptin-stimulated phosphorylation of hormone-sensitive lipase and consequent lipolysis, as do knockouts of dopamine β-hydroxylase, an enzyme required for catecholamine synthesis. Thus, neuro-adipose junctions are necessary and sufficient for the induction of lipolysis in white adipose tissue and are an efferent effector of leptin action. Direct activation of sympathetic inputs to adipose tissues may represent an alternative approach to induce fat loss, circumventing central leptin resistance. PAPERCLIP. 

Sympathetic neuro-adipose connections mediate leptin-driven lipolysis.

To become metastatic, a tumor cell must acquire new adhesion properties that allow migration into the surrounding connective tissue, transmigration across endothelial cells to reach the blood stream and, at the site of metastasis, adhesion to endothelial cells and transmigration to colonize a new tissue. Hydrogen peroxide (H2O2) is a redox signaling molecule produced in tumor cell microenvironment with high relevance for tumor development. However, the molecular mechanisms regulated by H2O2 in tumor cells are still poorly known. The identification of H2O2-target proteins in tumor cells and the understanding of their role in tumor cell adhesion are essential for the development of novel redox-based therapies for cancer. In this paper, we identified Ribosomal Protein SA (RPSA) as a target of H2O2 and showed that RPSA in the oxidized state accumulates in clusters that contain specific adhesion molecules. Furthermore, we showed that RPSA oxidation improves cell adhesion efficiency to laminin in vitro and promotes cell extravasation in vivo. Our results unravel a new mechanism for H2O2-dependent modulation of cell adhesion properties and identify RPSA as the H2O2 sensor in this process. This work indicates that high levels of RPSA expression might confer a selective advantage to tumor cells in an oxidative environment. 

Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA.

Helicobacter pullorum is an avian enterohepatic species that, more recently, has also been found as a naturally acquired infection in mice and rats, and isolated from patients with gastrointestinal and hepatobiliary diseases. In this work, the interaction between H. pullorum and murine macrophages was examined. Firstly, the impact of nitric oxide, which is an antimicrobial produced by mammalian macrophages, on H. pullorum 6350-92 viability and morphology was studied by colony-forming assays and light microscopy, respectively. Exposure to nitric oxide lowered H. pullorum viability, in a growth-phase-dependent manner, and decreased the mean cell size. However, the number of coccoid forms remained low, contrasting with what has been observed for other Helicobacter species. Confocal microscopy showed that H. pullorum is internalized by murine macrophages, triggering nitric oxide production that promotes phagocytosis and killing of the pathogen. Interaction between H. pullorum and macrophages stimulated secretion of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and MIP-2. These results show that H. pullorum is able to infect mammalian murine cells triggering an inflammatory response. 

Helicobacter pullorum induces nitric oxide release in murine macrophages that promotes phagocytosis and killing.

Marine bioadhesives have unmatched performances in wet environments, being an inspiration for biomedical applications. In sea urchins specialized adhesive organs, tube feet, mediate reversible adhesion, being composed by a disc, producing adhesive and de-adhesive secretions, and a motile stem. After tube foot detachment, the secreted adhesive remains bound to the substratum as a footprint. Sea urchin adhesive is composed by proteins and sugars, but so far only one protein, Nectin, was shown to be over-expressed as a transcript in tube feet discs, suggesting its involvement in sea urchin adhesion. Here we use high-resolution quantitative mass-spectrometry to perform the first study combining the analysis of the differential proteome of an adhesive organ, with the proteome of its secreted adhesive. This strategy allowed us to identify 163 highly over-expressed disc proteins, specifically involved in sea urchin reversible adhesion; to find that 70% of the secreted adhesive components fall within five protein groups, involved in exocytosis and microbial protection; and to provide evidences that Nectin is not only highly expressed in tube feet discs but is an actual component of the adhesive. These results give an unprecedented insight into the molecular mechanisms underlying sea urchin adhesion, and opening new doors to develop wet-reliable, reversible, and ecological biomimetic adhesives.

Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: A quantitative proteomics approach.

Batrachoidids, which include midshipman and toadfish are less known among embryologists, but are common in other fields. They are characteristic for their acoustic communication, and develop hearing and sound production while young juveniles. They lay large benthic eggs (>5mm) with a thick chorion and adhesive disk and slow development, which are particularly challenging for studying embryology. Here we took advantage of a classical tissue clearing technique and the OPenT open-source platform for optical tomography imaging, to image a series of embryos and larvae from 3 to 30mm in length, which allowed detailed 3D anatomical reconstructions non-destructively. We documented some of the developmental stages (early and late in development) and the anatomy of the delicate stato-acoustic organs, swimming bladder and associated sonic muscles. Compared to other techniques accessible to developmental biology labs, OPenT provided advantages in terms of image quality, cost of operation and data throughput, allowing identification and quantitative morphometrics of organs in larvae, earlier and with higher accuracy than is possible with other imaging techniques.

Optical micro-tomography "OPenT" allows the study of large toadfish Halobatrachus didactylus embryos and larvae.

Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc) versus the non-adhesive part (the stem), and also to profile the proteome of the secreted adhesive (glue). This data article contains complementary figures and results related to the research article "Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach" (Lebesgue et al., 2016) [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold), likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives. 

Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive.

Flow cytometry is the tool of choice for high-speed acquisition and analysis of large cell populations, with the tradeoff of lacking intracellular spatial information. Although in the last decades flow cytometry systems that can actually acquire two-dimensional spatial information were developed, some of the limitations remained though, namely constrains related to sample size and lack of depth or dynamic information. The combination of fluidics and light-sheet illumination has the potential to address these limitations. By having cells travelling with the flowing sheath one can, in a controlled fashion, force them at constant speed through the light-sheet enabling the synchronized acquisition of several optical sections, that is, three-dimensional imaging. This approach has already been used for imaging cellular spheroids, plankton, and zebra-fish embryos. In this review, we discuss the known solutions and standing challenges of performing three-dimensional high-throughput imaging of multicellular biological models using fluidics, while retaining cell and organelle-level resolution. © 2017 International Society for Advancement of Cytometry.

Three-dimensional imaging flow cytometry through light-sheet fluorescence microscopy.

Flatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.

A thyroid hormone regulated asymmetric responsive centre is correlated with eye migration during flatfish metamorphosis.

Formation of the vertebrate postcranial body axis follows two sequential but distinct phases. The first phase generates pre-sacral structures (the so-called primary body) through the activity of the primitive streak on axial progenitors within the epiblast. The embryo then switches to generate the secondary body (post-sacral structures), which depends on axial progenitors in the tail bud. Here we show that the mammalian tail bud is generated through an independent functional developmental module, concurrent but functionally different from that generating the primary body. This module is triggered by convergent Tgfbr1 and Snai1 activities that promote an incomplete epithelial to mesenchymal transition on a subset of epiblast axial progenitors. This EMT is functionally different from that coordinated by the primitive streak, as it does not lead to mesodermal differentiation but brings axial progenitors into a transitory state, keeping their progenitor activity to drive further axial body extension.

A Tgfbr1/Snai1-dependent developmental module at the core of vertebrate axial elongation.

During early embryogenesis, the hemogenic endothelium of the developing dorsal aorta is the main source of definitive hematopoietic stem cells (HSCs), which will generate all blood cell lineages of the adult organism. The hemogenic endothelial cells (HECs) of the dorsal aorta are known to arise from the splanchnic lateral plate mesoderm. However, the specific cell lineages and developmental paths that give rise to aortic HECs are still unclear. Over the past half a century, the scientific debate on the origin of aortic HECs and HSCs has largely focused on two potential and apparently alternative birthplaces, the extraembryonic yolk sac blood islands and the intraembryonic splanchnic mesoderm. However, as we argue, both yolk sac blood islands and aortic HECs may have a common hemangioblastic origin. Further insight into aortic HEC development is being gained from fate-mapping studies that address the identity of progenitor cell lineages, rather than their physical location within the developing embryo. In this perspective article, we discuss the current knowledge on the origin of aortic HECs with a particular focus on the evidence provided by studies in the avian embryo, a model that pioneered the field of developmental hematopoiesis.

Gabriel G. Martins

Instituto Gulbenkian de Ciência

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation