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

Analysis of opo cis-regulatory landscape uncovers Vsx2 requirement in early eye morphogenesis

The self-organized morphogenesis of the vertebrate optic cup entails coupling the activation of the retinal gene regulatory network to the constriction-driven infolding of the retinal epithelium. Yet the genetic mechanisms underlying this coordination remain largely unexplored. Through phylogenetic footprinting and transgenesis in zebrafish, here we examine the cis-regulatory landscape of opo, an endocytosis regulator essential for eye morphogenesis. Among the different conserved enhancers identified, we isolate a single retina-specific element (H6_10137) and show that its activity depends on binding sites for the retinal determinant Vsx2. Gain- and loss-of-function experiments and ChIP analyses reveal that Vsx2 regulates opo expression through direct binding to this retinal enhancer. Furthermore, we show that vsx2 knockdown impairs the primary optic cup folding. These data support a model by which vsx2, operating through the effector gene opo, acts as a central transcriptional node that coordinates neural retina patterning and optic cup invagination in zebrafish.

Characterization of the cis‐regulatory regions governing ojoplano locus

Ojoplano (opo) is a vertebrate-specific gene that was first identified in medaka fish as a recessive mutant, showing both neural crest defects and a failure of optic cup folding. In humans, this gene is associated with genetic diseases including hereditary craniofacial malformations and schizophrenia. It is localized in a 2Mb gene desert flanked by insulator sequences, between the genes SLC35B and TFAp2a. This region, syntenic between all vertebrates, represents only 2% of chromosome 6. However, it includes 23% of the all conserved cis-regulatory elements in this chromosome. Using transgenesis assays in zebrafish, we screened the enhancer activity of this locus and obtain a collection of nine enhancers. These regulatory elements were all conserved from human to teleosts and showed epigenetic marks for enhancer activity. We could associate multiple enhancers with ororfacial celfting disease and in order to explore the functionality of the enhancers, we performed a bioinformatics analysis to search for transcription factor bindings in the enhancer sequences. In terms of gene regulation we observe that H6:10137 opo enhancer has two Vsx2 binding sites and that this transcription factor regulates the expression of opo during eye development. Our findings suggest that the regulation of Vsx2 over opo is essential for optic cup folding. So far, there is no clear connection between optic cup patterning and morphogenesis. Vsx2 provides this link by controlling the expression of opo.

Role of insulin and insulin-like peptides in bone formation: identification of bone-specific target genes and regulatory mechanisms, and characterization of the insulin-mimetic effect of vanadium

Tese de Doutoramento em Biologia, Especialidade em Biologia Molecular, Universidade do Algarve, 2008

Chick heart/hemangioblast progenitor 12 (chep 12: study of a novel gene expressed in the heart/hemangioblast percursors cells

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009

Análise e caracterização da expressão e papel funcional dos potenciais genes da família 2 GPCRs no protostómio Caenorhabditis elegans

Dissertação de Mestrado, Biotecnologia, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009

Electro-olfactograms in response to chromatographic fractions of food-related odorants in the Senegalese sole, Solea senegalensis

The Bohr effect, which can be most generally defined as the influence of pH on the oxygen binding affinity of proteins, is a common feature of respiratory pigments, ranging from the haemocyanins of molluscs and crustaceans to the haemoglobins of vertebrates. Its physiological role is generally seen in the facilitation of oxygen release from respiratory pigments during tissue acidosis. The magnitude of the effect can be influenced by a multitude of factors such as temperature, carbon dioxide, chloride ions, organic phosphates and the investigated pH range. Here we present data on the maximal alkaline Bohr effect in haemoglobins from a large number of species covering all vertebrate classes, obtained at physiological temperatures in the presence of 100 mM chloride ions and the absence of carbon dioxide and organic phosphates.

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50–55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the ‘‘classical’’ role of pituitary function regulation.

11-ketotestosterone inhibits the alternative mating tactic in sneaker males of the peacock blenny, Salaria pavo

In the peacock blenny, Salaria pavo, a species with courtship sex-role reversal, smaller, younger males mimic the courtship behavior and the nuptial coloration of females in order to get access to nests during spawning and to parasitize egg fertilization from nest-holder males. Later in their life, sneakers transform both morphologically and behaviorally into nest-holder males. In the present paper we investigate the activational role of 11-ketotestosterone (KT), the most potent androgen in most teleost species, to promote the switch between tactics in sneaker males of S. pavo. Sneakers were implanted either with KT or with control (i.e. castor oil) silastic implants. A week after implantation they were subjected to a set of behavioral tests and morphometric measurements. KT treatment promoted the differentiation of secondary sex characters, such as the anal glands, and inhibited the expression of female courtship behavior. KT-treated sneakers also showed a trend toward less frequent display of female nuptial coloration. There was no effect of KT treatment on the expression of typical nest-holder male behavior. Finally, there was no effect of KT treatment on the number or soma size of arginine vasotocin neurons in the preoptic area, which are often associated with the expression of vertebrate sexual behavior. Thus, KT seems to play a key role in mating tactic switching by inhibiting the expression of female courtship behavior and by promoting the development of male displaying traits (e.g. anal glands). The lack of a KT effect on behavior typical of nest-holding males and vasotocinergic preoptic neurons suggests that a longer time frame or other endocrine/social signals are needed for the initiation of these traits in males that are switching tactics.

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.

Evolution of matrix and bone gamma-carboxyglutamic acid proteins in vertebrates

The evolution of calcified tissues is a defining feature in vertebrate evolution. Investigating the evolution of proteins involved in tissue calcification should help elucidate how calcified tissues have evolved. The purpose of this study was to collect and compare sequences of matrix and bone γ-carboxyglutamic acid proteins (MGP and BGP, respectively) to identify common features and determine the evolutionary relationship between MGP and BGP. Thirteen cDNAs and genes were cloned using standard methods or reconstructed through the use of comparative genomics and data mining. These sequences were compared with available annotated sequences (a total of 48 complete or nearly complete sequences, 28 BGPs and 20 MGPs) have been identified across 32 different species (representing most classes of vertebrates), and evolutionarily conserved features in both MGP and BGP were analyzed using bioinformatic tools and the Tree-Puzzle software. We propose that: 1) MGP and BGP genes originated from two genome duplications that occurred around 500 and 400 million years ago before jawless and jawed fish evolved, respectively; 2) MGP appeared first concomitantly with the emergence of cartilaginous structures, and BGP appeared thereafter along with bony structures; and 3) BGP derives from MGP. We also propose a highly specific pattern definition for the Gla domain of BGP and MGP. Previous Section Next Section BGP1 (bone Gla protein or osteocalcin) and MGP (matrix Gla protein) belong to the growing family of vitamin K-dependent (VKD) proteins, the members of which are involved in a broad range of biological functions such as skeletogenesis and bone maintenance (BGP and MGP), hemostasis (prothrombin, clotting factors VII, IX, and X, and proteins C, S, and Z), growth control (gas6), and potentially signal transduction (proline-rich Gla proteins 1 and 2). VKD proteins are characterized by the presence of several Gla residues resulting from the post-translational vitamin K-dependent γ-carboxylation of specific glutamates, through which they can bind to calcium-containing mineral such as hydroxyapatite. To date, VKD proteins have only been clearly identified in vertebrates (1) although the presence of a γ-glutamyl carboxylase has been reported in the fruit fly Drosophila melanogaster (2) and in marine snails belonging to the genus Conus (3). Gla residues have also been found in neuropeptides from Conus venoms (4), suggesting a wider prevalence of γ-carboxylation.

Identification and study of novel genes expressed in the heart precursor cells

Understanding heart development on a molecular level is a requirement for uncovering the causes of congenital heart diseases. Several genes have been implicated as critical for heart development. However, the inducers of these genes as well as their targets and pathways, remain largely unknown. We have identified a promoter element of chick cCer able to drive EGFP expression in a population of cells that consistently exit from the anterior primitive streak region, from as early as stage HH3+, and that later will populate the heart. Using this promoter element as a tool allowed us to identify novel genes previously not known to potentially play a role in heart development. In order to identify and study genes expressed and involved in the correct development and differentiation of the vertebrate heart precursor cell (HPC) lineages, a differential screening using Affymetrix GeneChip® system technologies was performed. Remarkably, this screening led to the identification of more than 700 transcripts differentially expressed in the heart forming regions (HFR). Bioinformatic tools allowed us to filter the large amount of data generated from this approach and to select a few transcripts for in vivo validation. Five genes were selected for further characterization by whole mount in situ hybridization leading to the validation of their expression in the HPC. From those, Adtk1 and Ccbe1 were selected for functional analysis. Regarding to ccbe1, a more detailed WISH analysis was performed and showed that Ccbe1 is expressed specifically on the cardiac progenitors regions at HH4, more specifically in primary heart field and at later stages is present in the second heart field. Further functional analyses by knockdown and overexpression revealed an important role for Ccbe1 in early heart tube formation. Moreover, the results presented in this thesis suggested that Ccbe1 is a key gene during heart development and might be limited to multipotent and highly proliferative progenitors and downregulated upon cellular commitment into more specific cardiac phenotypes. Other of the genes identified, Adtk1 was also subjected to further functional studies. Knockdown of Adtk1 using morpholino oligonucleotides suggested that it might be necessary for the migration and fusion of the heart tube as well as for neural tube closure.

Evolution, gene regulation and functional analysis of BMP2 in fish

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.

Molecular characterization of Gla-rich protein (GRP) gene expression and function

Gla-rich protein (GRP) is a vitamin K-dependent protein related to bone and cartilage recently described. This protein is characterized by a large number of Gla (γ-carboxyglutamic acid) residues being the protein with the highest Gla content of any known protein. It was found in a widely variety of tissues but highest levels was found in skeletal and cartilaginous tissues. This small secreted protein was also expressed and accumulated in soft tissues and it was clearly associated with calcification pathologies in the same tissues. Although the biological importance of GRP remains to be elucidated, it was suggested a physiological role in cartilage development and calcification process during vertebrate skeleton formation. Using zebrafish, an accepted model to study skeletal development, we have described two grp paralog genes, grp1 and grp2, which exhibited distinct patterns of expression, suggesting different regulatory pathways for each gene. Gene synteny analysis showed that grp2 gene is more closely related to tetrapod grp, although grp1 gene was proposed to be the vertebrate ortholog by sequence comparison. In addition, we identified a functional promoter of grp2 gene and using a functional approach we confirmed the involvement of transcription factors from Sox family (Sox9b and Sox10) in the regulation of grp2 expression. In an effort to provide more information about the function of grp isoforms, we generated two zebrafish transgenic lines capable to overexpress conditionally grp genes and possible roles in the skeleton development were studied. To better understand GRP function a mammalian system was used and the analysis of knockout mice showed that GRP is involved in chondrocyte maturation and the absence of GRP is associated to proteoglycans loss in calcified articular cartilage. In addition, we detected differences in chondrogenesis markers in articular chondrocyte primary culture. Overall, our data suggest a main role for GRP on chondrocyte differentiation.