Co-localization of GnRH ligands and their receptors in the European sea bass, Dicentrarchus labrax

The migration of the hypophysiotropic GnRH (GnRH-I) neurons during early development is a crucial step in establishing a normally functioning reproductive system in all vertebrates. These neurons derive from progenitor cells in the olfactory placode and subsequently migrate to their final position in the ventral forebrain, where they mediate hypophysiotropic control over Lh. We use zebrafish as a model to investigate the path and the factors that mediate the migration of the GnRH-I neurons during early development. A transgenic line of zebrafish, in which GnRH- I neurons specifically express a reporter gene (GFP) has been developed in our lab. This was achieved by integrating a GnRH-I promoter/GFP reporter transgene into the zebrafish genome. The resulting transgenic line allows us to track the route of the GnRH-I neuronal migration in real time and in vivo. We have used this line to conduct time lapse imaging to ascertain the exact migrational path and the final position in the ventral forebrain of the GnRH-I neurons.

Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes

Whole animal studies have indicated that Ca2+ uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using aminoterminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of 125I-(1–35tyr) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (KD=2.59 nM; Bmax=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1–34) PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The aminoterminal peptides (2–34)PTHrP, (3–34)PTHrP and (7–34) PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1–34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca2+ uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.

Insights into molecular and cellular events involved in normal and pathological vertebral development and fusion using zebrafish as model organism

The vertebral column and its units, the vertebrae, are fundamental features, characteristic of all vertebrates. Developmental segregation of the vertebral bodies as articulated units is an intrinsic requirement to guarantee the proper function of the spine. Whenever these units become fused either during development or postsegmentation, movement is affected in a more or less severe manner, depending on the number of vertebrae affected. Nevertheless, fusion may occur as part of regular development and as a physiological requirement, like in the tetrapod sacrum or in fish posterior vertebrae forming the urostyle. In order to meet the main objective of this PhD project, which aimed to better understand the molecular and cellular events underlying vertebral fusion under physiological and pathological conditions, a detailed characterization of the vertebral fusion occurring in zebrafish caudal fin region was conducted. This showed that fusion in the caudal fin region comprised 5 vertebral bodies, from which, only fusion between [PU1++U1] and ural2 [U2+] was still traceable during development. This involved bone deposition around the notochord sheath while fusion within the remaining vertebral bodies occur at the level of the notochord sheath, as during the early establishment of the vertebral bodies. A comparison approach between the caudal fin vertebrae and the remaining vertebral column showed conserved features such as the presence of mineralization related proteins as Osteocalcin were identified throughout the vertebral column, independently on the mineralization patterns. This unexpected presence of Osteocalcin in notochord sheath, here identified as Oc1, suggested that this gene, opposing to Oc2, generally associated with bone formation and mature osteoblast activity, is potentially associated with early mineralization events including chordacentrum formation. Nevertheless, major differences between caudal fin region and anterior vertebral bodies considering arch histology and mineralization patterns, led us to use RA as an inductive factor for vertebral fusion, allowing a direct comparison of equivalent structures under normal and fusion events. This fusion phenotype was associated with notochord sheath ectopic mineralization instead of ectopic perichordal bone formation related with increased osteoblast activity, as suggested in previous reports. Additionally, alterations in ECM content, cell adhesion and blood coagulation were discussed as potentially related with the fusion phenotype. Finally, Matrix gla protein, upregulated upon RA treatment and shown to be associated with chordacentrum mineralization sites in regular development, was further described considering its potential function in vertebral formation and pathological fusion. Therefore with this work we propose zebrafish caudal fin vertebral fusion as a potential model to study both congenital and postsegmentation fusion and we present candidate factors and genes that may be further explored in order to clarify whether we can prevent vertebral fusion.

Identification and molecular characterization of bone-related micrornas: functional implications

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Effects of vitamin K deficiency and its mechanisms in vertebrate's early development: zebrafish as a model

Disertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

From embryonic stem cells to sensory hair cells : a programming approach

Tese de doutoramento, Ciências Biomédicas (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Medicina, 2014

Das perceções das crianças sobre alguns animais mal-afamados à sua (re) construção em contexto de sala de aula

Relatório de Estágio apresentado à Escola Superior de Educação de Lisboa para obtenção do grau de mestre em Ensino do 1.º e 2.º Ciclo do Ensino Básico

Palaeontological data about the climatic trends from Chattian to present along the Northeastern Atlantic frontage

Climatic changes that affected the Northeastern Atlantic frontage are analyzed on the basis of the evolution of faunas and floras from the late Oligocene onwards. The study deals with calcareous nannoplankton, marine micro- and macrofaunas, some terrestrial vertebrates and vegetal assemblages. The climate, first tropical, underwent a progressive cooling (North-South thermic gradient). Notable climatic deteriorations (withdrawal towards the South or disappearance of taxa indicative of warm climate and appearance of "cold" taxa) are evidenced mainly during the Middle Miocene and the late Pliocene. Faunas and floras of modern pattern have regained, after the Pleistocene glaciations, a new climatic ranging of a temperate type in the northern part.

Bipedal Locomotion: A Fractional CPG for Generating Patterns

Proceedings of the 10th Conference on Dynamical Systems Theory and Applications

Extension and patterning of the vertebrate body

Dissertation presented to obrain the Ph.D degree in Biology. Developmental Biology

Hematopoiesis in the Drosophila larva: beyond the lymph gland

All coelomate animals possess a population of cells that do not make part of an organ and instead freely flow inside the body cavity. These cells, termed hemocytes (in invertebrates) or blood cells (in vertebrates), are involved in varied functions including immune response, clearance of apoptotic cells and distribution of nutrient and gases (Grigorian & Hartenstein 2013).(...)

Size-Control Regulation during Regeneration

Functional regeneration of organs upon injury is a key process for animals survival. Contrary to humans, some vertebrates are remarkably competent in regenerating after acute organ or appendage lesions. This advantageous skill allows overcoming limitations in repair even in adult stages, when tissues are fully developed, via a process of epimorphic regeneration. One such organism is the zebrafish, which can regenerate several organs, namely its heart, retina, spinal cord and fins. (...)

Fate map of zebrafish left-right organizer

The organizer is a ciliated signalling transient organ, responsible for the patterning of embryo tissues during embryonic development. In higher vertebrates, such as mouse and chick, this organizer (the node and the Hensen’s node, respectively) performs dorsalventral and anteriorposterior axis definition, as well as left-right patterning of the internal organs. In lower vertebrates, such as frog and zebrafish, there is a separate specialized organ for left-right purposes called the Gastrocoel Roof Plate (GRP) and Kupffer’s Vesicle (KV), respectively. It is known that mouse and chick organizer cells give rise to structures like floor plate, notochord, hypochord and somites. Frog GRP originates all these but floor plate. In zebrafish, at 13-14 somite stage (ss) the KV finished its left-right patterning but what happens to this organizer’ cells is still poorly studied. This research attempts to understand the fate and behaviour of the KV cells. We followed the fate of KV cells by live imaging and by tight time-courses with fixed larvae. We assessed in detail their proliferative and death profile, as well as cilia length progression from 9-10 ss until 29-30 ss. We conclude that the KV cells mostly follow the evolutionarily conserved fates described for other organizers. These cells mainly incorporate the notochord and hypochord; few cells incorporate the floor plate and the somites. As a novelty, it is also hypothesized that the hypural cell fate may be among the KV cell fates.

Linking melanism to brain development: expression of a melanism-related gene in barn owl feather follicles covaries with sleep ontogeny.

BACKGROUND: Intra-specific variation in melanocyte pigmentation, common in the animal kingdom, has caught the eye of naturalists and biologists for centuries. In vertebrates, dark, eumelanin pigmentation is often genetically determined and associated with various behavioral and physiological traits, suggesting that the genes involved in melanism have far reaching pleiotropic effects. The mechanisms linking these traits remain poorly understood, and the potential involvement of developmental processes occurring in the brain early in life has not been investigated. We examined the ontogeny of rapid eye movement (REM) sleep, a state involved in brain development, in a wild population of barn owls (Tyto alba) exhibiting inter-individual variation in melanism and covarying traits. In addition to sleep, we measured melanistic feather spots and the expression of a gene in the feather follicles implicated in melanism (PCSK2). RESULTS: As in mammals, REM sleep declined with age across a period of brain development in owlets. In addition, inter-individual variation in REM sleep around this developmental trajectory was predicted by variation in PCSK2 expression in the feather follicles, with individuals expressing higher levels exhibiting a more precocial pattern characterized by less REM sleep. Finally, PCSK2 expression was positively correlated with feather spotting. CONCLUSIONS: We demonstrate that the pace of brain development, as reflected in age-related changes in REM sleep, covaries with the peripheral activation of the melanocortin system. Given its role in brain development, variation in nestling REM sleep may lead to variation in adult brain organization, and thereby contribute to the behavioral and physiological differences observed between adults expressing different degrees of melanism.

Facies, depositional environment, and palaeoecology of the Middle Triassic Cassina Beds (Meride Limestone, Monte San Giorgio, Switzerland).

The Ladinian Cassina beds belong to the fossiliferous levels of the world-famous Middle Triassic Monte San Giorgio Lagerstatte (UNESCO World Heritage List, Canton Ticino, Southern Alps). Although they are a rich archive for the depositional environment of an important thanatocoenosis, previous excavations focused on vertebrates and particularly on marine reptiles. In 2006, the Museo Cantonale di Storia Naturale (Lugano) started a new research project focusing for the first time on microfacies, micropalaeontological, palaeoecological and taphonomic analyses. So far, the upper third of the sequence has been excavated on a surface of around 40 m(2), and these new data complete those derived from new vertebrate finds (mainly fishes belonging to Saurichthys, Archaeosemionotus, Eosemionotus and Peltopleurus), allowing a better characterization of the basin. Background sedimentation on an anoxic to episodically suboxic seafloor resulted in a finely laminated succession of black shales and limestones, bearing a quasi-anaerobic biofacies, which is characterized by a monotypic benthic foraminiferal meiofauna and has been documented for the first time from the whole Monte San Giorgio sequence. Event deposition, testified by turbidites and volcaniclastic layers, is related to sediment input from basin margins and to distant volcanic eruptions, respectively. Fossil nekton points to an environment with only limited connection to the open sea. Terrestrial macroflora remains document the presence of emerged areas covered with vegetation and probably located relatively far away. Proliferation of benthic microbial mats is inferred on the basis of microfabrics, ecological considerations and taphonomic (both biostratinomic and diagenetic) features of the new vertebrate finds, whose excellent preservation is ascribed to sealing by biofilms. The occurrence of allochthonous elements allows an insight into the shallow-waters of the adjoining time-equivalent Salvatore platform. Finally, the available biostratigraphic data are critically reviewed.