Differential expression of notch signaling-related transcripts accompanies Pro-thymocyte proliferation and phenotype transition induced by epidermal growth factor plus insulin in fetal thymus organ cultures

Thymus regression upon stressing stimuli, such as infectious diseases, is followed by organ reconstitution, paralleling its development in ontogeny. A narrow window of thymus development was here studied, encompassing the pro-T lymphoid precursor expansion during specification stages, by the use of epidermal growth factor plus insulin (INS) in murine fetal thymus organ cultures. Aiming to disclose signaling pathways related to these stages, cultured thymus lobes had their RNA extracted, for the search of transcripts differentially expressed using RNAse protection assays and reverse transcriptase-polymerase chain reactions. We found no difference that could explain INS-driven thymocyte growth, in the pattern of transcripts for death/proliferation mediators, or for a series of growth factor receptors and transcriptional regulators known as essential for thymus development. Thymocyte suspensions from cultured lobes, stained for phenotype analysis by fluorescence activated cell sorting, showed a decreased staining for Notch1 protein at cell surfaces upon INS addition. We analyzed the expression of Notch-related elements, and observed the recruitment of a specific set of transcripts simultaneous and compatible with INS-driven thymocyte growth, namely, transcripts for Notch3, for its ligand Jagged2, and for Deltex1, a mediator of a poorly characterized alternative pathway downstream of the Notch receptor.

Fator de crescimento epidérmico na ontogenia do timo murino

O fator de crescimento epidérmico (EGF) foi usado em cultivo de timo fetal murino, para que se testasse seu possível efeito na ontogenia deste órgão. EGF, na dose de 20 ng/ml (aproximadamente 3, 2nM) ou acima, bloqueou o desenvolvimento dos compartimentos linfóide e microambiental do timo, afetando o crescimento e a diferenciação de timócitos. Foi especialmente atingido o componente linfóide majoritário (TCR[gama][BETA]+) em um momento crítico, na transição de um fenótipo duplo-negativo (CD4-8-; DN) para duplo-positivo (CD4+8+; DP). Tal efeito foi mimetizado por inibidores da função tirosina-quinase (tirfostinas) específicos para o receptor de EGF (EGF-R), e parcialmente revertido por insulina, no que diz respeito ao crescimento mas não à diferenciação de timócitos. Foi observada em timócitos fetais, assim como em DN adultos, uma molécula de superfície imunorreativa para anti-soro anti-EGF murino, ausente nas células remanescentes em cultivo na presença de EGF. Tais achados apontam para um novo circuito biológico relacionado à ontogenia do timo, envolvendo o EGF-R expresso por células do microambiente e a molécula "EGF-imunorreativa" de timócitos.

Epidermal growth factor (EGF) was added to fetal thymus cultures, aiming to assess a putative effect on the ontogeny ofthis organ. At doses over 20 ng/ml (approximately 3.2 nM), the soluble factor affected both the development of lymphoid and microenvironmental compartments, with a blockade of thymocyte growth and differentiation. The major thymocyte subset (TCRab+) was particularly affected, at the critical point that represents the DN ® DP transition (CD4-8- ® CD4+8-). This effect was mimicked by inhibitors of tyrosine-kinase function (tyrphostins) specific for the EGF receptor (EGF-R), and partially reverted by insulin, as regards thymocyte growth but not differentiation. Fetal thymocytes, as well as adult DN thymocytes, were revealed to bear an "EGFimmunoreactive" surface molecule, detected with an anti-EGF antiserum, and that was absent on those thymocytes remaining in cultures in the presence of EGF. Taken together our data point to the existence of a novel biological circuit related to the thymus ontogeny involving the micro environmental EGF-R and the thymocyte "EGF-immunoreactive" molecule.