Juvenile Hormone Suppresses The Selection Of Sensory Organ Precursors To Block Drosophila Adult Abdominal Bristle Formation

Juvenile hormone（JH）is a key hormone in insect metamorphosis,which is mainly responsible for maintaining larval growth and preventing metamorphosis of larvae to pupae and adults,with a typical"status quo"effect.Early studies found that exogenous application of a juvenile-preserving hormone analogue during white pre-pupae in Drosophila melanogaster could inhibit the formation of abdominal bristle.Although significant progress has been made on the mechanism of action of JH in inhibiting insect metamorphosis,little research has been reported on the molecular mechanism that prevents the morphogenesis of abdominal bristle in Drosophila adults.Studies have shown that abdominal bristle formation in Drosophila adults mainly undergoes three morphogenetic events namely proliferation,migration and differentiation of histoblast.In this study,studies were initiated from the effects of JH on these three physiological events,with a view to elucidating the regulatory mechanism of JH on the formation of abdominal bristles in Drosophila adults and providing further theoretical basis for the in-depth study of the physiological function of JH.The specific findings are summarized below:In this experiment,we firstly used Esg-Gal4,UAS-GFP,tub-Gal80^ts/cyo;TM2/TB strains to specifically label abdominal histoblast with green fluorescence by Drosophila GAL4/UAS and GAL80^ts genetic techniques,and analyzed the proliferation and migration of histoblast by observing the distribution of green fluorescence.The results showed that there was no significant difference in the fluorescence intensity and fluorescence area between the JH treated group and the control group（acetone-treated）at 0-12 h after puparium formation（APF）,which is the rapid proliferation stage of histoblast in Drosophila.Meanwhile,the transcript expression level of string gene,which is responsible for the G2/M transition of cytokinesis,was detected by q RT-PCR.The results showed that string m RNA expression had no significant difference between the two groups.These results suggest that JH does not affect the proliferation of histoblast.Further,to investigate the effect of JH on the migration of histoblast,the migration of adult tissue cells at 12-40 h APF was observed and analyzed.Statistics showed that there was no significant difference between the control group and the treatment group,indicating that JH did not affect the migration of histoblast.To investigate whether JH has an effect on the differentiation of adult tissue cells to produce sensory organ precursor（SOP）cells,immunofluorescence techniques were used to indicate SOP cells by Cut protein and neu A101-lac Z Drosophila strain,respectively,while q RT-PCR was used to detect the specific expression of genes in SOP.The transcript levels of senseless were investigated for SOP formation in abdominal histoblast.The results showed that at 20 h APF,the beginning of differentiation of histoblast,there was significant expression of Cut in the control group,but not detected in the treated group,and the number of positive cells for Cut in the treated group was always lower than that in the control group as development progressed;and the transcript level of senseless was significantly lower in the treated group.Meanwhile,the lac Z immunofluorescence results of the neu A101-lac Z Drosophila strain were consistent with the Cut immunofluorescence results.The above results indicated that JH inhibited the differentiation of histoblast into SOP.Given that the number of SOP was closely related to the expression of proneuronal genes achaete（ac）and scute（sc）,the changes of ac and sc expression at 12-24 h APF stage were detected using q RT-PCR,and the results showed that the levels of ac and sc m RNA were significantly decreased at 15-22 h.The above results suggest that JH inhibits SOP formation by suppressing the expression of proto-neural genes,which ultimately inhibits the formation of abdominal setae in Drosophila adults.Notch signaling can block the expression of ac and sc through the transcriptional blocker E（spl）-HLH family.The target gene E（spl）m3-HLH in the Notch signaling pathway was detected using q RT-PCR to characterize Notch activity.The results showed that the expression level of E（spl）m3-HLH,a direct transcriptional target of Notch,was significantly increased in the abdomen of JHM-treated worms at 18 h APF and 22 h APF,indicating that JH analogue treatment suppressed the expression of the proneuronal genes ac and sc by activating Notch activity,which in turn led to a reduction in the number of SOP in the abdomen of JH analogue-treated Drosophila.Finally,we explored whether the JH primary response gene Kruppel-homolog1（Kr-h1）mediated the inhibitory effect of JH on SOP formation.Using the GAL4/UAS system to specifically overexpress or RNAi reduce Kr-h1 expression in histoblast cells,we found that Kr-h1 overexpression had effects similar to those of JH treatment,a decrease in the number of adult abdominal setae,a decrease in the number of SOP,a decrease in ac and sc transcript expression levels,and an increase in E（spl）m3-HLH expression.In contrast,when Kr-h1 was knocked down the effect of JH was correspondingly attenuated.The above results suggest that the inhibitory effect of JH on bristle formation is mainly mediated through Kr-h1.In conclusion,we determined that JH inhibits the formation of abdominal SOP in Drosophila melanogaster through Kr-h1,thus affecting the formation of abdominal bristle.This study is the first to investigate the molecular mechanism of JH inhibition of bristle formation,and provides a theoretical basis for the study of the mechanism of JH“status quo”action.