FUNCTIONAL ANALYSIS OF CADHERINS IN Apis mellifera: PRODUCTION OF SPECIFIC ANTIBODIES AND RNAi MEDIATED SILENCING OF GENE FUNCTION

• Grant number: 07/05416-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2008
• End date: July 31, 2011
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Klaus Hartmann Hartfelder
• Grantee: Mônica Mazzei Florecki
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 05/03926-5 - Functional genomics of Apis mellifera: search for new genes and functional networks in the context of development, different castes and reproduction, AP.TEM

Abstract

Honeybees are highly social insects characterized by the overlap of generations and by the presence of sexes and castes phenotipically distinct. Drones and queens are responsible for the reproductive process, whereas workers are functionally sterile and perform different tasks throughout their lives. The ovary of Apis mellifera is classified as meroistic polytrophic. In this system, oogenesis initiates with the asymmetric division of a germline stem cell that generates a new stem cell and a cystoblast. Each cystoblast undergoes a series of incomplete mitotic divisions to form a cyst of germ cells that will differentiate subsequently into an oocyte and several nurse cells. Spermatogenesis is also initiated by the division of a germline stem cell, called primary spermatogonia, that generates another stem cell and a secondary spermatogonia which undergoes a series of mitotic divisions before entering meiosis. Studies have demonstrated the importance of cadherins in invertebrate gametogenesis, mainly in Drosophila, where E-cadherin is necessary for anchoring germline stem cells to the top of the testioles and ovarioles. In ovaries and testes of Apis mellifera, cadherins are dispersed throughout the cytoplasm of germ and somatic cells which indicates that cadherins may have additional roles besides cellular adhesion. However, as the experiments to localize cadherins in the gonads of Apis mellifera were performed with heterologous antibodies against the cytoplasmic domain of chicken N-cadherin, we cannot be certain about the types of cadherins they recognize. For this reason, the aim of this project is to produce antibodies against sequences of extracellular and cytoplasmic domains of E-cadherin and N-cadherin of Apis mellifera, which should permit us to study the expression of each one and verify whether the cadherins which we previously visualized in the cytoplasm of germ and somatic cells are in fact whole proteins or just the cleaved cytoplasmic domains. Functional studies will be performed by dsRNA-mediated silencing of E-cadherin and N-cadherin gene function. In this approach, ovaries and testes will be maintained in culture medium and subsequently analyzed by RT-PCR, Western blot, immunofluorescence and possibly also a honey bee genomic microarray. Antibody production together with gene silencing should contribute to clarify the function(s) of cadherins in the gonads and add relevant information to the morphological analyses of honey bee gametogenesis performed so far.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

FLORECKI, MONICA M.; HARTFELDER, KLAUS. Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads. Cell Biology International, v. 35, n. 1, p. 45-49, JAN 2011. (07/05416-0)