In vivo characterization of two novel regulators of the JAK/STAT pathway in Drosophila melanogaster
KAUSTIO, MERI (2011)
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KAUSTIO, MERI
2011
Biokemia - Biochemistry
Biolääketieteellisen teknologian yksikkö - Institute of Biomedical Technology
Hyväksymispäivämäärä
2011-11-04Tiivistelmä
Background and aims: The JAK/STAT pathway is a well-conserved signalling pathway that takes part in many developmental and functional processes in both humans and flies. In both organisms, defects in this pathway have been found to result in the development of pathological conditions such as cancer and immune diseases. Therefore, there is great interest in identifying new genes that take part in the regulation of signalling through the JAK/STAT pathway. Drosophila melanogaster is a well-suited model organism for screening and studying new regulators of JAK/STAT signalling, as unlike mammals, it lacks redundancy in the key components of the pathway. The aim of this project was to study the effect of overexpression of two genes, et and not4, found in an RNAi screen for new JAK/STAT pathway regulators in Drosophila, and to verify their function as JAK/STAT pathway regulators.
Methods: The UAS-GAL4 system was used for directed gene expression in Drosophila melanogaster in vivo. UAS-not4 and UAS-et transgenic lines were obtained from the Best Gene Inc. and balanced against suitable balancers. Balanced lines were crossed over the drivers C564-GAL4 or eyeless-GAL4 to study the effects of not4 and et overexpression on the stress response or eye-development of the fly in vivo, respectively. Gene expression was assayed with quantitative RT-PCR.
Results: Overexpression of not4 in the fatbody of the fly caused an increase in the expression of the stress response genes TotA and TotM, which are known JAK/STAT pathway targets. not4 expression was, however, not induced by septic injury, which is known to activate JAK/STAT signalling. et expression in turn was strongly induced by septic injury. Despite previous results, et overexpression caused only mild changes in the expression of Tot genes and could not prevent their induction following septic injury. However, as has also been previously shown elsewhere, et knock-down caused dramatic upregulation of Tot gene expression. Overexpression of not4 in the eye-discs had no visible effect on the development or morphology of the fly eye.
Conclusions: Based on previous knowledge, Et seems to function as a negative regulator of JAK/STAT signalling, but in our assay the overexpression of et could not prevent the transcription of pathway targets in vivo. In any case, since the expression of et is induced by septic injury, Et seems to be involved in the stress response of Drosophila. not4 overexpression evidently increases the expression of the JAK/STAT targets TotA and TotM, which in conjunction with evidence from other experiments suggests that Not4 functions as a positive regulator of JAK/STAT signalling. However, the mechanisms of function of both Not4 and Et still require further studying.
Methods: The UAS-GAL4 system was used for directed gene expression in Drosophila melanogaster in vivo. UAS-not4 and UAS-et transgenic lines were obtained from the Best Gene Inc. and balanced against suitable balancers. Balanced lines were crossed over the drivers C564-GAL4 or eyeless-GAL4 to study the effects of not4 and et overexpression on the stress response or eye-development of the fly in vivo, respectively. Gene expression was assayed with quantitative RT-PCR.
Results: Overexpression of not4 in the fatbody of the fly caused an increase in the expression of the stress response genes TotA and TotM, which are known JAK/STAT pathway targets. not4 expression was, however, not induced by septic injury, which is known to activate JAK/STAT signalling. et expression in turn was strongly induced by septic injury. Despite previous results, et overexpression caused only mild changes in the expression of Tot genes and could not prevent their induction following septic injury. However, as has also been previously shown elsewhere, et knock-down caused dramatic upregulation of Tot gene expression. Overexpression of not4 in the eye-discs had no visible effect on the development or morphology of the fly eye.
Conclusions: Based on previous knowledge, Et seems to function as a negative regulator of JAK/STAT signalling, but in our assay the overexpression of et could not prevent the transcription of pathway targets in vivo. In any case, since the expression of et is induced by septic injury, Et seems to be involved in the stress response of Drosophila. not4 overexpression evidently increases the expression of the JAK/STAT targets TotA and TotM, which in conjunction with evidence from other experiments suggests that Not4 functions as a positive regulator of JAK/STAT signalling. However, the mechanisms of function of both Not4 and Et still require further studying.