Biochemistry and Molecular Biology of Development

Summary

The face of vertebrates presents a dazzling array of shapes and sizes, depending largely on the development of the craniofacial skeleton. In particular, cartilage and bones of the face develop from a specific population of neural crest cells. These cells form a series of pharyngeal arches almost identical in all vertebrates. The question then is how these cells are organized in facial features appropriate for each animal? This question is fundamental to understanding not only how diversity is generated faces of vertebrates, but also how they originate in human congenital craniofacial defects affecting development of the face.

In our laboratory we develop two research lines trying to answer questions related to the development of craniofacial structures. The approach is at two levels: the modeling of human craniofacial disorders (cleft palate, aberrations in the formation of maxilla and mandible, etc.) employing the zebrafish as experimental model, and the characterization of molecular mechanisms that control gene expression by involving single-stranded nucleic acid binding proteins.

We use technologies of gain- and loss-of-function of specific proteins (loss of function by microinjection of morpholinos or dominant negative protein overexpression and gain of function by overexpression of wild protein), generation of transgenic fish lines, as well various techniques of developmental biology. In addition, molecular biology techniques, biochemistry and spectroscopy (absorption, fluorescence, and circular dichroism) are usually employed.

As a complementary activity, and derived from knowledge about the biology of zebrafish, in the laboratory it is carried out the DarT test as a fast, simple and highly sensitive and reproducible method to assess water and effluent treatment quality and/or toxicity of synthetic or natural compounds. The test is provided as a service to monitor in water, industrial effluents and sediments, the presence of compounds potentially toxic to humans or the ecosystem.

Research Lines

Modelling human craniofacial pathologies in Zebrafish

The zebrafish has numerous advantages when modelling human diseases. It is a simple model, small, economical, fast-growing and highly fertile. Using this model, we try to explain the molecular basis of human craniofacial disorders which etiology is still not fully understood. We generated a model of Treacher Collins syndrome and identified a group of genes that are currently being studied to establish
their biological roles and molecular relationships. Similarly, we are modelling other human craniofacial disorders to identify and characterize their molecular basis. Craniofacial structures derive primarily from the neural crest, so we try to identify which neural crest marker genes modify their expression during the disease and, what stage of neural

Control of gene expression through molecular folding of guanine quadruplex

The success of embryo development depends on the spatiotemporal control of gene expression, which involves proteins interacting with nucleic acids. Among these, single-stranded nucleic acids binding proteins are versatile actors in the control of gene expression as they may act in different cellular processes. Single-stranded Guanine-rich regions present in DNA or RNA of can fold adopting a tertiary structure named Guanine-quadruplex or G4, which may regulate gene expression. We study CNBP, a protein essential for specification and differentiation of craniofacial structures. CNBP specifically recognizes G-rich single-stranded DNA and, acting as nucleic acid chaperone,
modulates G4 formation in promoter regions of specific genes. Our studies are aimed to the identification of the CNBP cellular targets,
as well as the molecular mechanisms by which this protein performs its biological role.

Selected Publications

  • Melo US, Macedo-Souza LI, Figueiredo T, Muotri AR, Gleeson JG, Coux G, Armas P, Calcaterra NB, Kitajima JP, Amorim S, Olávio TR, Griesi-Oliveira K, Coatti GC, Rocha CR, Martins-Pinheiro M, Menck CF, Zaki MS, Kok F, Zatz M, Santos S (2015). Overexpression of KLC2 due to a homozygous deletion in the non-coding region causes SPOAN syndrome. Hum Mol Genet. 24(24): 6877-6885. DOI: 10.1093/hmg/ddv388
  • David AP, Margarit E, Domizi P, Banchio C, Armas P, Calcaterra NB (2016). G-quadruplexes as novel cis-elements controlling transcription during embryonic development. Nucleic Acids Res. 44: 4163-4173. DOI: 10.1093/nar/gkw011
  • Porcel de Peralta MS, Mouguelar VS, Sdrigotti MA, Ishiy F, Fanganiello RD, Passos-Bueno MR, Coux G, Calcaterra NB. (2016). Cnbp ameliorates Treacher Collins Syndrome craniofacial anomalies through a pathway that involves redox-responsive genes. Cell Death & Disease, 7: e2397. DOI: 10.1038/cddis.2016.299.
  • Armas P, David A, Calcaterra NB (2017). Transcriptional control by G-quadruplexes: in vivo roles and perspectives for specific intervention. Transcription., 8: 21-25. DOI: 10.1080/21541264.2016.1243505.
  • Armas P, Calcaterra NB (2018). G-quadruplex in animal development: Contribution to gene expression and genomic heterogeneity. Mech Dev. 154: 64-72. DOI: 10.1016/j.mod.2018.05.004
  • Weiner AMJ (2018). MicroRNAs and the neural crest: From induction to differentiation. Mech Dev. 154: 98-106. DOI: 10.1016/j.mod.2018.05.00
  • Rosas MG, Lorenzatti A, Porcel de Peralta MS, Calcaterra NB, Coux G (2019). Proteasomal inhibition attenuates craniofacial malformations in a zebrafish model of Treacher Collins Syndrome. Biochem Pharmacol. 163: 362-370. DOI: 1016/j.bcp.2019.03.005
  • Weiner AMJ, Scampoli NL, Steeman TJ, Dooley CM, Busch-Nentwich EM, Kelsh RN, Calcaterra NB (2019). Dicer1 is required for pigment cell and craniofacial development in zebrafish. Biochim Biophys Acta Gene Regul Mech. 1862(4): 472-485. DOI: 10.1016/j.bbagrm.2019.02.005
  • David AP, Pipier A, Pascutti F, Binolfi A, J Weiner AM, Challier E, Heckel S, Calsou P, Gomez D, Calcaterra NB, Armas P (2019). CNBP controls transcription by unfolding DNA G-quadruplex structures. Nucleic Acids Res. 47(15): 7901-7913. DOI: 10.1093/nar/gkz527
  • Cedron VP, Weiner AMJ, Vera M, Sanchez, L (2020). Acetaminophen affects the survivor, pigmentation and development of craniofacial structures in zebrafish (Danio rerio) embryos. Biochemical Pharmacology 174: 113816. DOI: 10.1016/j.bcp.2020.113816
  • Weiner AMJ, Coux, G, Armas, P, Calcaterra N (2021). Insights into vertebrate head development: from cranial neural crest to the modelling of neurocristopathies. Int J Dev Biol. 65: 215-225. DOI: 10.1387/ijdb.200229nc
  • Bezzi G, Piga E, Binolfi A, Armas P (2021). CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands. Int. J. Mol. Sci. 22(5): 2614. DOI: 10.3390/ijms22052614
  • Steeman TJ, Rubiolo JA, Sánchez LE, Calcaterra NB, Weiner AMJ (2021). Conservation of Zebrafish MicroRNA-145 and Its Role during Neural Crest Cell Development. Genes (Basel) 12(7): 1023. DOI: 10.3390/genes12071023
  • Armas P, Coux G, Weiner AMJ, Calcaterra N (2021). What's new about CNBP? Divergent functions and activities for a conserved nucleic acid binding protein. Biochimica et Biophysica Acta (BBA) - General Subjects. In press. DOI: 10.1016/j.bbagen.2021.129996

Collaborators

  • Néstor Carrillo. Laboratorio de Biología del Estrés en Plantas, IBR - CONICET-UNR. Rosario, Argentina.
  • Andrés Binolfi. Laboratorio de Biología Estructural-Celular, IBR - CONICET-UNR. Rosario, Argentina.
  • Julia Cricco. Laboratorio de Biología y Bioquímica de Trypanosoma cruzi, IBR - CONICET-UNR. Rosario, Argentina.
  • Sebastián Rius. CEFOBI - CONICET-UNR. Rosario, Argentina.
  • María Rita Passos-Bueno, Laboratorio de Genética del Desarrollo Humano, Departamento de Genética y Biología Evolutiva - Instituto de Biociencias, Universidad de San Pablo. San Pablo, Brasil.
  • Juan Pablo Nicola, CIBICI (Centro de Investigación en Bioquímica Clínica e Inmunología) CCT CONICET – Córdoba. Córdoba, Argentina.
  • Laura Sánchez y Juan Rubiolo, Facultad de Veterinarias, Universidad de Santiago de Compostela. Lugo, España.
  • Robert Kelsh, Department of Biology and Biochemistry, University of Bath. Bath, Reino Unido.
  • Dennis Gomez-Zamorano. Institut de Pharmacologie et Biologie Structurale (IPBS) – CNRS. Toulouse, Francia.

Grants

  • PIP-CONICET 2015-0170 (IR: Nora Calcaterra).
  • PICT-2016-0671, Tipo A (IR: Nora Calcaterra).
  • PICT 2016-0367, Tipo D (IR: Gabriela Coux).
  • Investigación Orientada 2018 – ASACTeI, MinCyT, Provincia de Santa Fe - IO 2018-000296 (IR: Gabriela Coux).
  • PICT-2017-0507, Tipo B (IR: Andrea Weiner).
  • PICT-2017-0976, Tipo A (IR: Pablo Armas).
  • Investigación Aplicada en PyMEs 2018 IBR-CIBIC-HERITAS. – ASaCTeI, MinCyT, Provincia de Santa Fe - IA-2018-0075 (IR: Nora Calcaterra).
  • 3 Subsidios BIO / UNR. IR: Pablo Armas, Nora Calcaterra y Gabriela Coux.
  • PICT-2019-1662, Tipo A (IR: Pablo Armas).
  • PICT 2019-0307, Tipo D (IR: Gabriela Coux).
  • PICT 2019-0763, Tipo D (IR: Andrea Weiner).

Director de Grupo

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Calcaterra, Nora B.
Core CCT
Email: calcaterra@ibr-conicet.gov.ar
Phone: +54 341 4237070
Office Extension: 655
Laboratory Extension: 614