Study On The Mechanisms Of Transcription Factor Apontic Regulating The Development Of Neuron And Wing In Drosophila

Neuroblast 6-4(NB6-4)is a perfect system to elucidate the mechanism of neural differentiation.The thoracic neuroblast 6-4(NB6-4T)gives rise to three glial cells and four to six interneurons,while the abdominal NB6-4(NB6-4A)gives rise to only two glial cells.To date,the mechanism governing the distinct differentiation of NB6-4A and NB6-4T is still unclear.Drosophila is a perfect model to study the development process.We use the embryo nervous system and larval wing discs to study the function of transcription factor Apontic(Apt).Apt binds to DNA through a 14 bp palindrome sequence ATTCCAATTGGAAT to activate the expression of target genes,such as cyclinE(cycE)and hedgehog(hh).Previous studies have identified Apt as an important factor to regulate the development of tracheae,head,heart,eye and border cell.In addition,FSBP,which is a human homolog of Apt,regulates the development of lung,liver,skeletal muscle,kidney,and pancreas.FSBP plays a critical role in the cancer.However,the function of Apt in the development of nervous system and wing discs is not well known.CycE is an important factor which regulates S phase entry and cell fate determination of neural cells.CycE plays a critical role in the NB6-4T neural precursor cells and promotes the neural precursor differentiate into 4-6 neural cells.While Abd-A and Abd-B suppress the expression of cycE in the NB6-4A,resulting in differentiation into only two glial cells.However,the regulation mechanism of Cyc E during neural differentiation is still unclear.Glial cell missing(Gcm)is a switch,which controls the differentiation of glial cells.Gcm protein is a transcription factor and determines glial cells fate versus neural cells fate by regulating the expression of glia-specific genes.Gcm controls the glial cells differentiation except the midline glial cells.Gcm expresses in the glial part of NB6-4T and NB6-4A.The first cell division of NB6-4T is asymmetric,giving rise to a glial precursor cell and a neural precursor cell.Pros controls the expression of gcm in the glial precursor cell,and the glial precursor cell differentiates into glial cells.The neural precursor cell differentiates into 4-6neural cells without the expression of gcm.However,the downstream events of Gcm are still unclear.Notch signaling pathway is evolutionally conserved and involved in multiple cellular processes,such as cell division,cell fate determination,cell death.Without the ligand,the Notch signaling pathway is closed.In the presence of the ligand Delta(Dl),Notch is cleaved into two parts to release the C-terminal Notch intra-cellular domain(NICD).In turn,NICD enters into the nucleus to turn on the expression of target genes.For this perspective,it functions directly in signal transduction through complexing with the CBF1/RBPjk/Su(H)/Lag1(CSL)DNA binding protein and transcriptional coactivators to switch on expression of Notch target genes.The study of Notch is also of particular interest to neural biologists,as Notch mediates lateral inhibition.The signaling out of the signaling cell results in differentiating into the neural cells.In contrast,the signaling receiving cells are prohibited to process differentiation.However,the previous studies focused on studying the downstream genes regulation of Notch signaling pathway and function analysis,the regulation of Notch upstream genes remains to be clarified.Herein,using genetic experiments,we figured out that Apt maintained the cell fate of neural cells through activating cycE expression.Immunostaining results showed that apt exclusively in NB6-4T cells,not in NB6-4A cells.Loss of apt resulted in neural cell lost,which was rescued,at least in part by overexpressing cycE.On the other hand,we uncovered that Gcm suppressed apt expression in NB6-4 glial cells.Collectively,our findings suggest that Apt is a potent master regulator to control neural cell fate maintenance.In addition,we revealed that both knockdown of and overexpression of apt produced deformed wings,indicating that Apt regulates wing development.Mechanistically,apt genetically interacts with Notch signaling pathway ligand delta.Apt affects protein level of Delta.Apt activities Notch signal pathway and its target gene wg.The functional analysis of Apt could provide a theoretical basis for understanding the mechanism of nervous system development,wing disc development,and Notch signaling pathway activation.The main results presented in this thesis are as follows:(1)Apt controls the differentiation of neural cells through activating the expression of cycE during the development of nervous systemWe marked NB6-4 cells with Eg staining.Immunostaining analyses revealed that Apt and Eg co-localized in the NB6-4T cells.This result suggests that apt expresses in the NB6-4T cells.apt mutant embryos diminished the expression of cycE during NB6-4T development,culminating in loss of the NB6-4T neural cells in stage 10 and stage 12.In contrast,overexpression of apt led to the ectopic neural cells in the NB6-4A.Furthermore,neural cell lost induced by apt mutant was effectively restored by ectopic expressing cycE,indicating that apt sits upstream of cycE to modulate neural cell fate maintenance.(2)Gcm controls the differentiation of glial cells through repressing the expression of apt and cycE during the development of nervous systemLoss of gcm resulted in glial precursor lost.Immunostaining analyses demonstrated that Gcm suppresses apt and cycE expression in the NB6-4.Via RT-qPCR assays,the suppressive effects of Gcm upon apt and cycE was validated by overexpressing gcm.In addition,the glial cells restarted differentiation in the double mutant of gcm and apt embryo.Taken together,these results suggest that Gcm is important to suppress the expression of apt and cycE.(3)Apt activates Notch signaling pathway in the development of wing discsKnockdown of apt resulted in a samller and notched wing,indicating that Apt is likely involved in Notch pathway regulation.Our Microarray and RT-qPCR data showed that Apt activated the expression of neur,which encodes an activator of Notch signaling pathway.Furthermore,epistasis assays showed that apt genetically interacted with delta.Knockdown of apt decreased the protein levels of Delta,NICD and Wg in the wing disc.On contrary,overexpression of apt resulted in ectopic expression of Delta,NICD and Wg.Based on Wg-lacZ reporter experiment,we illustrated that Apt promoted the transcription of wg.Taken together,Apt activates Notch signalling possibly through promoting neur expression.We will dissect the detail mechanisms in the future.