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Researcher Department of Molecular Biology and Genetics
Ana Paula Azambuja and Marcos Simoes-Costa
Developmental Cell, ISSN: 15345807, eISSN: 18781551, Pages: 1268-1282.e6, Published: 3 May 2021 Elsevier BV
Cell fate commitment is controlled by cis-regulatory elements often located in remote regions of the genome. To examine the role of long-range DNA interactions in early development, we generated a high-resolution contact map of active enhancers in avian neural crest cells. This analysis uncovered a diverse repertoire of enhancers that are part of the gene regulatory network underlying specification. We found that neural crest identity is largely regulated by cis-regulatory elements that propagate signaling inputs to network components. These genomic sensors display a combination of optimal and suboptimal TCF/LEF-binding sites, which allow cells to respond to Wnt signaling in a position-dependent manner. We propose that, rather than acting as upstream activators, signaling systems feed into regulatory circuits in a hub-and-spoke architecture. These results shed light on the tridimensional organization of the neural crest genome and define how signaling systems provide progenitors with spatial cues that transform their molecular identity.
Ana Paula Azambuja and Marcos Simoes-Costa
PLoS Genetics, ISSN: 15537390, eISSN: 15537404, Published: 19 January 2021 Public Library of Science (PLoS)
The process of cell fate commitment involves sequential changes in the gene expression profiles of embryonic progenitors. This is exemplified in the development of the neural crest, a migratory stem cell population derived from the ectoderm of vertebrate embryos. During neural crest formation, cells transition through distinct transcriptional states in a stepwise manner. The mechanisms underpinning these shifts in cell identity are still poorly understood. Here we employ enhancer analysis to identify a genetic sub-circuit that controls developmental transitions in the nascent neural crest. This sub-circuit links Wnt target genes in an incoherent feedforward loop that controls the sequential activation of genes in the neural crest lineage. By examining the cis-regulatory apparatus of Wnt effector gene AXUD1, we found that multipotency factor SP5 directly promotes neural plate border identity, while inhibiting premature expression of specification genes. Our results highlight the importance of repressive interactions in the neural crest gene regulatory network and illustrate how genes activated by the same upstream signal become temporally segregated during progressive fate restriction.
Debadrita Bhattacharya, Ana Paula Azambuja, and Marcos Simoes-Costa
Developmental Cell, ISSN: 15345807, eISSN: 18781551, Pages: 199-211.e6, Published: 20 April 2020 Elsevier BV
The Warburg effect is one of the metabolic hallmarks of cancer cells, characterized by enhanced glycolysis even under aerobic conditions. This physiological adaptation is associated with metastasis , but we still have a superficial understanding of how it affects cellular processes during embryonic development. Here we report that the neural crest, a migratory stem cell population in vertebrate embryos, undergoes an extensive metabolic remodeling to engage in aerobic glycolysis prior to delamination. This increase in glycolytic flux promotes Yap/Tead signaling, which activates the expression of a set of transcription factors to drive epithelial-to-mesenchymal transition. Our results demonstrate how shifts in carbon metabolism can trigger the gene regulatory circuits that control complex cell behaviors. These findings support the hypothesis that the Warburg effect is a precisely regulated developmental mechanism that is anomalously reactivated during tumorigenesis and metastasis.
Ana Paula Azambuja and Marcos Simoes-Costa
Methods in Molecular Biology, ISSN: 10643745, Volume: 1920, Pages: 99-110, Published: 2019 Springer New York
The chick embryo is a powerful model for experimental embryology due to its accessibility, sturdiness, and ease of manipulation. Here we describe protocols for analysis of protein-DNA and protein-protein interactions in tissues and cells isolated from the developing chick. These assays are aimed at the identification of interactions between transcription factors and regulatory elements in the genome, and, in combination with functional assays, can be used for the delineation of gene regulatory circuits.
Debadrita Bhattacharya, Megan Rothstein, Ana Paula Azambuja, and Marcos Simoes-Costa
eLife, eISSN: 2050084X, Published: 1 December 2018 eLife Sciences Publications, Ltd
A crucial step in cell differentiation is the silencing of developmental programs underlying multipotency. While much is known about how lineage-specific genes are activated to generate distinct cell types, the mechanisms driving suppression of stemness are far less understood. To address this, we examined the regulation of the transcriptional network that maintains progenitor identity in avian neural crest cells. Our results show that a regulatory circuit formed by Wnt, Lin28a and let-7 miRNAs controls the deployment and the subsequent silencing of the multipotency program in a position-dependent manner. Transition from multipotency to differentiation is determined by the topological relationship between the migratory cells and the dorsal neural tube, which acts as a Wnt-producing stem cell niche. Our findings highlight a mechanism that rapidly silences complex regulatory programs, and elucidate how transcriptional networks respond to positional information during cell differentiation.
Alexander R. Moise, Ângela M.S. Costa, Murilo Carvalho, Ana P. Azambuja, José Xavier-Neto, and Hozana A. Castillo
Endothelium and Cardiovascular Diseases: Vascular Biology and Clinical Syndromes, Pages: 11-22, Published: 19 January 2018 Elsevier
Abstract We discuss coronary ontogeny and function in an evolutionary framework. Coronary morphogenesis is old, appearing at the level of the first jawed vertebrates. After its origins, the presence or absence of coronaries seems to be dictated more in response to physical activity than due to phylogenetic origin. The major coronary vessels derive from the proepicardium, which is a cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells. Here we review the basic notion that coronary development is a highly organized process, in which coronary endothelial differentiation and tubular morphogenesis precedes coronary smooth muscle cell differentiation and incorporation into true coronary vessels. Some years ago, we showed that these complex steps are orchestrated by retinoic acid and the vascular endothelial growth factor. We also examine new views on the origins of coronary vessels (i.e., the sinus venosus) and explore the potential of the epicardium in coronary and cardiac regeneration.
Aline Fagundes Martins, José Xavier Neto, Ana Azambuja, Maria Lorena Sereno, Antonio Figueira, Paulo Henrique Campos-Junior, Millor Fernandes Rosário, Cristiane Bittencourt Barroso Toledo, Gerluza Aparecida Borges Silva, Gregory Thomas Kitten, Luiz Lehmann Coutinho, Susanne Dietrich, and Erika Cristina Jorge
Cells Tissues Organs, ISSN: 14226405, eISSN: 14226421, Volume: 200, Pages: 326-338, Published: 4 November 2015 S. Karger AG
Repulsive guidance molecules (RGMs) compose a family of glycosylphosphatidylinositol (GPI)-anchored axon guidance molecules and perform several functions during neural development. New evidence has suggested possible new roles for these axon guidance molecules during skeletal muscle development, which has not been investigated thus far. In the present study, we show that RGMa, RGMb and RGMc are all induced during skeletal muscle differentiation in vitro. Immunolocalization performed on adult skeletal muscle cells revealed that RGMa, RGMb and RGMc are sarcolemmal proteins. Additionally, RGMa was found to be a sarcoplasmic protein with a surprisingly striated pattern. RGMa colocalization with known sarcoplasmic proteins suggested that this axon guidance molecule is a skeletal muscle sarcoplasmic protein. Western blot analysis revealed two RGMa fragments of 60 and 33 kDa, respectively, in adult skeletal muscle samples. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa overexpression produced hypertrophic cells, whereas RGMa knockdown resulted in the opposite phenotype. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types. Our results are the first to show an axon guidance molecule as a skeletal muscle sarcoplasmic protein and to include RGMa in a system that regulates skeletal muscle cell size and differentiation.
Ana Paula De Oliveira Azambuja, Edna Regina Netto-Oliveira, Amauri Aparecido Bássoli de Oliveira, Maximiliano Dos Anjos Azambuja, and Wilson Rinaldi
Acta Scientiarum - Health Sciences, ISSN: 16799291, eISSN: 18078648, Pages: 59-64, Published: 2014 Universidade Estadual de Maringa
Cardiovascular diseases are the main cause of death in world population. Current analysis with 1074 schoolchildren in municipal schools of Cruzeiro do Oeste, Parana State, Brazil, evaluated anthropometric indexes and blood pressure levels of schoolchildren and verified the relationship between Body Mass Index (BMI) and Waist Circumference (WC) with blood pressure (BP). Evaluations comprised weight and height for the calculation of BMI, waist circumference and arterial pressure. Chi-square test and Cramer's coefficient verified whether the variables were associated. High percentage of weight excess was reported, or rather, 16.6% of children were overweight; 9.8% were obese; 30.6% had high WC and 38.3% had high blood pressure. BMI and WC were associated with high BP. Overweight schoolchildren with central adiposity had a great trend of having high blood pressure.
Ana Paula de O. Azambuja, Edna Regina Netto-Oliveira, Amauri Aparecido B. de Oliveira, Maximiliano dos Anjos Azambuja, and Wilson Rinaldi
Revista Paulista de Pediatria, ISSN: 01030582, Pages: 166-171, Published: June 2013 FapUNIFESP (SciELO)
OBJETIVO: Associar o estado nutricional com sexo, grupo etário e nível econômico de escolares do ensino fundamental por meio de um estudo transversal nas escolas municipais do município de Cruzeiro do Oeste, Paraná, Brasil. MÉTODOS: A pesquisa foi realizada em 2010 com 939 alunos com idades entre seis e dez anos. Para tanto, realizou-se a classificação do nível econômico das famílias por meio do questionário de ranking econômico (Associação Brasileira de Empresas de Pesquisa) e foi calculado o índice de massa corpórea. A análise dos dados foi realizada por estatística descritiva, e a verificação de diferenças entre grupos independentes pelos testes de Mann-Whitney e Kruskal-Wallis, bem como possíveis associações foram verificadas pelo teste do qui-quadrado. Para todos os procedimentos adotou-se o nível de significância de p<0,05. RESULTADOS: A prevalência de obesidade na amostra estudada foi de 8,0% e de sobrepeso, 16,4%. Não houve associação entre nível econômico e estado nutricional, assim como entre estado nutricional e sexo. CONCLUSÕES: A prevalência de sobrepeso e obesidade foi considerada alta, evidenciando que crianças de ambos os sexos são afetadas, independentemente de seus níveis econômicos e grupo etário.
José Xavier-Neto, Brad Davidson, Marcos Sawada Simoes-Costa, Rodrigo Abe Castro, Hozana Andrade Castillo, Allysson Coelho Sampaio, and Ana Paula Azambuja
Heart Development and Regeneration, Pages: 3-45, Published: 2010 Elsevier
Publisher Summary This chapter discusses various kinds of animal circulatory pumps and utilizes a hierarchical set of concepts to analyze their evolution. It also defines the position occupied by vertebrate chambered hearts in relation to the other classes of animal pumping organs, such as arthropod dorsal vessels and mollusc chambered hearts, traces the origins of these diverse pumping organs, focuses on the evolution of deuterostome pumping organs, and discusses evolutionary scenarios regarding the origins of vertebrate hearts. The comparative study of pumping organs in bilaterian animals is rife with provoking and stimulating similarities that in some cases reflect true homologies. In other cases, however, the distinction is not yet possible, but it is helpful to note that the requisites for homology are very well known today, and that it is necessary to check the available evidence against these criteria. However, similarities between animal pumping organs need not be homologies to be interesting. In many cases, these similarities can be conveniently attributed to evolutionary parallelisms or to convergence (Hall, 2003; Xavier-Neto et al., 2007). These parallel or convergent solutions to the problems of animal pumping are, in a sense, actually more challenging than the cases of homology. The evolution of chambered hearts from the simple peristaltic organs of invertebrate chordates remains a fascinating problem whose evolutionary, developmental and genetic bases have been left largely untouched.
Ana P. Azambuja, Victor Portillo-Sánchez, Mariliza V. Rodrigues, Samantha V. Omae, Deborah Schechtman, Bryan E. Strauss, Eugenia Costanzi-Strauss, José E. Krieger, José M. Perez-Pomares, and José Xavier-Neto
Circulation Research, ISSN: 00097330, eISSN: 15244571, Volume: 107, Pages: 204-216, Published: 23 July 2010 Ovid Technologies (Wolters Kluwer Health)
Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels.
H. A. Castillo, R. M. Cravo, A. P. Azambuja, M. S. Simoes-Costa, S. Sura-Trueba, J. Gonzalez, E. Slonimsky, K. Almeida, J. G. Abreu, M. A. A. de Almeida, T. P. Sobreira, S. H. P. de Oliveira, P. S. L. de Oliveira, I. A. Signore, A. Colombo, M. L. Concha, T. S. Spengler, M. Bronner-Fraser, M. Nobrega, N. Rosenthal, and J. Xavier-Neto
Development, ISSN: 09501991, eISSN: 14779129, Volume: 137, Pages: 507-518, Published: 1 February 2010 The Company of Biologists
Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.
Marcos S. Simões-costa, Ana Paula Azambuja, and José Xavier-Neto
Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, ISSN: 15525007, eISSN: 15525015, Volume: 310, Pages: 54-72, Published: 15 January 2008 Wiley
Signaling by retinoic acid (RA) is an important pathway in the development and homeostasis of vertebrate and invertebrate chordates, with a critical role in mesoderm patterning. Classical studies on the distribution of nuclear receptors of animals suggested that the family of RA receptors (RARs/NR1B) was restricted to chordates, while the family of RA X receptors (RXR/NR2B) was distributed from cnidarians to chordates. However, the accumulation of data from genome projects and studies in non-model species is questioning this traditional view. Here we discuss the evidence for non-chordate RA signaling systems in the light of recent advances in our understanding of carotene (pro-Vitamin A) metabolism and of the identification of potential RARs and members of the NR1 family in echinoderms and lophotrochozoan trematodes, respectively. We conclude, as have others before (Bertrand et al., 2004. Mol Biol Evol 21(10):1923-1937), that signaling by RA is more likely an ancestral feature of bilaterians than a chordate innovation.
J. Xavier-Neto, R. A. Castro, A. C. Sampaio, A. P. Azambuja, H. A. Castillo, R. M. Cravo, and M. S. Simões-Costa
Cellular and Molecular Life Sciences, ISSN: 1420682X, eISSN: 15691632, Pages: 719-734, Published: March 2007 Springer Science and Business Media LLC
Research in animal models established that tinman, a key gene in Drosophila dorsal vessel development, is an orthologue of Nkx2-5, a key gene in vertebrate cardiac development. Similarities between the arthropod dorsal vessel and vertebrate hearts are interpreted in light of concepts such as homology or convergence. We discuss this controversy in the context of the evolution of animal circulatory pumps and propose the distinction between peristaltic and chambered pumps as a fundamental parameter for evolutionary comparisons between bilaterian pumps. Neither homology nor convergence is satisfactory to explain the origins of hearts and pumping organs. Instead, we propose that animal pumps derive from parallel improvements of an ancestral, peristaltic design represented by a layer of myocytes at the external walls of primitive vessels. This paradigm unifies disparate views, impacts our understanding of bilaterian evolution and may be helpful to interpret similarities between pumping organs of phylogenetically relevant species and emerging models.