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- Targeted Inactivation of Cerberus Like-2 Leads to Left Ventricular Cardiac Hyperplasia and Systolic Dysfunction in the MousePublication . Araújo, Ana Carolina; Marques, Sara; Belo, José A.Previous analysis of the Cerberus like 2 knockout (Cerl2(-/-)) mouse revealed a significant mortality during the first day after birth, mostly due to cardiac defects apparently associated with randomization of the left-right axis. We have however, identified Cerl2-associated cardiac defects, particularly a large increase in the left ventricular myocardial wall in neonates that cannot be explained by laterality abnormalities. Therefore, in order to access the endogenous role of Cerl2 in cardiogenesis, we analyzed the embryonic and neonatal hearts of Cerl2 null mutants that did not display a laterality phenotype. Neonatal mutants obtained from the compound mouse line Cer2(-/-)
- The activity of cerberus-like 2 during cardiogenesis, morphological and morphogenetics studiesPublication . Araújo, Ana Carolina; Belo, José A.
- Transthoracic echocardiography reference values in juvenile and adult 129/Sv micePublication . Vinhas, Maurícia; Araújo, Ana Carolina; Ribeiro, Sónia; Rosário, Luís Brás; Belo, José A.Background In the recent years, the use of Doppler-echocardiography has become a standard non-invasive technique in the analysis of cardiac malformations in genetically modified mice. Therefore, normal values have to be established for the most commonly used inbred strains in whose genetic background those mutations are generated. Here we provide reference values for transthoracic echocardiography measurements in juvenile (3 weeks) and adult (8 weeks) 129/Sv mice. Methods Echocardiographic measurements were performed using B-mode, M-mode and Doppler-mode in 15 juvenile (3 weeks) and 15 adult (8 weeks) mice, during isoflurane anesthesia. M-mode measurements variability of left ventricle (LV) was determined. Results Several echocardiographic measurements significantly differ between juvenile and adult mice. Most of these measurements are related with cardiac dimensions. All B-mode measurements were different between juveniles and adults (higher in the adults), except for fractional area change (FAC). Ejection fraction (EF) and fractional shortening (FS), calculated from M-mode parameters, do not differ between juvenile and adult mice. Stroke volume (SV) and cardiac output (CO) were significantly different between juvenile and adult mice. SV was 31.93 ± 8.67 μl in juveniles vs 70.61 ± 24.66 μl in adults, ρ < 0.001. CO was 12.06 ± 4.05 ml/min in juveniles vs 29.71 ± 10.13 ml/min in adults, ρ < 0.001. No difference was found in mitral valve (MV) and tricuspid valve (TV) related parameters between juvenile and adult mice. It was demonstrated that variability of M-mode measurements of LV is minimal. Conclusions This study suggests that differences in cardiac dimensions, as wells as in pulmonary and aorta outflow parameters, were found between juvenile and adult mice. However, mitral and tricuspid inflow parameters seem to be similar between 3 weeks and 8 weeks mice. The reference values established in this study would contribute as a basis to future studies in post-natal cardiovascular development and diagnosing cardiovascular disorders in genetically modified mouse mutant lines.
- A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencingPublication . Pereira-Leal, José B.; Abreu, Isabel A.; Alabaça, Cláudia S.; Almeida, Maria H.; Almeida, Paulo; Almeida, Tânia; Amorim, Maria I.; Araújo, Susana; Azevedo, Herlânder; Badia, Aleix; Batista, Dora; Bohn, Andreas; Capote, Tiago; Carrasquinho, Isabel; Chaves, Inês; Coelho, A. C.; Costa, Maria M. R.; Costa, Rita; Cravador, A.; Egas, Conceição; Faro, Carlos; Fortes, Ana M.; Fortunato, Ana S.; Gaspar, Maria J.; Gonçalves, Sónia; Graça, José; Horta, Marília; Inácio, Vera; Leitão, J. M.; Lino-Neto, Teresa; Marum, Liliana; Matos, José; Mendonça, Diogo; Miguel, Andreia; Miguel, Célia M.; Morais-Cecílio, Leonor; Neves, Isabel; Nóbrega, Filomena; Oliveira, Maria M.; Oliveira, Rute; Pais, Maria S.; Paiva, Jorge A.; Paulo, O. S.; Pinheiro, Miguel; Raimundo, João A. P.; Ramalho, J. C.; Ribeiro, Ana I.; Ribeiro, Teresa; Rocheta, Margarida; Rodrigues, Ana I.; Rodrigues, José C.; Saibo, Nelson J. M.; Santo, Tatiana; Santos, Ana M.; Sá-Pereira, Paula; Sebastiana, Mónica; Simões, Fernanda; Sobral, Rómulo S.; Tavares, Rui; Teixeira, Rita; Varela, Carolina; Veloso, Maria M.; Ricardo, Cândido P. P.Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.
- The activity of Mouse cerberus like 2 during cardiogenesis: genetic and morphogenetic studiesPublication . Araújo, Ana Carolina; Belo, José AntónioThe heart is the first organ that becomes functional in the vertebrate embryo. Heart morphogenesis is a complex process, with precise control developmental mechanisms, that can nevertheless fail. There are morphological aspects as polarity of the heart intrinsically related with the three body axes, anterior?posterior (A-P), dorsal?ventral (D-V), and left?right (L-R). The L-R axis has became subject of many studies in recent years and was found that the heart undergoes multiple morphogenetic processes, which are governed by this axis. Development of internal organs proceeds across the L-R axis and gain shape during organogenesis as a result of the early asymmetric activation of the conserved Nodal signalling cascade, in the left lateral plate mesoderm (Hamada et al., 2002). A Cerberus/Dan family member, mouse cerberus-like2 (cerl-2) is asymmetrically expressed on the right side of the mouse node and encodes for a secreted protein that binds directly to nodal restricting the Nodal signalling pathway towards the left side by preventing its activity in the right side (Marques et al, 2004). Preliminary studies showed that cerl-2 knockout (KO) mice display multiple laterality defects including heart?s rotation failure and randomization of organs? position due to L/R axis disruption. In addition, was observed several cardiac defects as severe hyperplasia of the myocardium and incomplete atria formation and ventricular septation that may not be explained by laterality abnormalities. In this study, were conducted morphological analyses of cerl-2 KO newborns, histological sections of newborn hearts and WISH with Gata-4, mefc2, hand and fgf8 probes on embryos throughout heart development (7,5dpc to 10.5dpc). Furthermore, a new compound mouse line cerl- 2KO::mlc1vlacZ was generated which will help to identify the contribution of the Secondary Heart Field (SHF) to the cerl-2KO heart defects. This body of work leads to the suggestion that, in addition to the previously described laterality-related defects, another distinct mechanism may contribute to the spectrum of complex cardiac defects in cerl-2 KO mice that cannot be explained only by the disruption of the nodal cascade in LPM. Problems in heart morphogenesis lead to congenital heart disease, which is the most common form of birth defect in humans (Harvey, 2002; Olson and Schenider, 2003). Abstract
- The activity of mouse Cerberus like 2 during cardiogenesis - genetic and morphogenetic studiesPublication . Araújo, Ana Carolina; Belo, José AntónioCardiogenesis is a delicate and complex process that requires the coordination of an intricate network of pathways and the different cell types. Therefore, understanding heart development at the morphogenetic level is an essential requirement to uncover the causes of congenital heart disease and to provide insight for disease therapies. Mouse Cerberus like 2 (Cerl2) has been defined as a Nodal antagonist in the node with an important role in the Left-Right (L/R) axis establishment, at the early embryonic development. As expected, Cerl2 knockout mice (Cerl2-/-) showed multiple laterality defects with associated cardiac failure. In order to identify the endogenous role of Cerl2 during heart formation independent of its described functions in the node, we accurately analyzed animals where laterality defects were not present. We thereby unravel the consequences of Cerl2 lossof- function in the heart, namely increased left ventricular thickness due to hyperplasia of cardiomyocytes and de-regulated expression of cardiac genes. Furthermore, the Cerl2 mutant neonates present impaired cardiac function. Once that the cardiac expression of Cerl2 is mostly observed in the left ventricle until around midgestration, this result suggest a specific regulatory role of Cerl2 during the formation of the left ventricular myoarchitecture. Here, we present two possible molecular mechanisms underlying the cardiac Cerl2 function, the regulation of Cerl2 antagonist in activation of the TGFßs/Nodal/Activin/Smad2 signaling identified by increased Smad2 phosphorilation in Cerl2-/- hearts and the negative feedback between Cerl2 and Wnt/ß-catenin signaling in heart formation. In this work and since embryonic stem cells derived from 129 mice strain is extensively used to produce targeted mutants, we also present echocardiographic reference values to progressive use of juveniles and young adult 129/Sv strain in cardiac studies. In addition, we investigate the cardiac physiology of the surviving Cerl2 mutants in 129/Sv background over time through a follow-up study using echocardiographic analysis. Our results revealed that Cerl2-/- mice are able to improve and maintain the diastolic and most of systolic cardiac physiologic parameters as analyzed until young adult age. Since Cerl2 is no longer expressed in the postnatal heart, we suggest that an intrinsic and compensatory mechanism of adaptation may be active for recovering the decreased cardiac function found in Cerl2 mutant neonates. Altogether, these data highlight the role of Cerl2 during embryonic heart development in mice. Furthermore, we also suggest that Cerl2-/- may be an interesting model to uncover the molecular, cellular and physiological mechanisms behind the improvement of the cardiac function, contributing to the development of therapeutic approaches to treat heart failures.