ZBTB20 Regulates Differentiation And Function Of Lactotropes In The Pituitary

Pituitary is the endocrine central organ of mammals,which plays an important role in maintaining individual growth,development,reproduction,metabolism,and homeostasis of internal environment.The anterior pituitary gland is mainly composed of five endocrine cells,including somatotropes,lactotropes,thyrotrophs,corticotrophs and gonadotrophs,among which the first three endocrine cells originate from PIT1-positive progenitors.Prolactin(PRL)secreted by lactotropes plays a key role in promoting mammary gland development,as well as stimulating and maintaining lactation.Lactotropes account for about 20%～50%of the total number of adult pituitary endocrine cells,which can be more than 50%in the pituitary of pregnant and lactating female mice.Prolactin tumor is the most common pituitary tumor in clinic.Therefore,to explore the molecular mechanism of specification and development of lactotropes and their homeostasis is not only of great scientific significance,but also of potential clinical value and application prospects.Mice model are commonly used to study pituitary development.The development of lactotrophs in mice was later than that of somatotropes,mainly originating from growth hormone(GH)cell lineages in middle and late stage of embryo.It is generally believed that somatolactotropes of GH+PRL+ are precursors to the early initiation of targeted specification of lactotrophs.Before and after birth,the double positive progenitor cells began to differentiate into GH-PRL+progenitor cells,which then further proliferate and differentiate into mature lactotrophs.But the double positive progenitor cells still maintained certain proliferative activity after birth.GH-PRL+progenitor cells undergo a rapid expansion period 2-3 weeks after birth.Previous studies in our laboratory have found that zinc finger protein ZBTB20 is a key molecule in regulating pituitary development and cell fate specification of lactotropes during early development.ZBTB20 was highly expressed in lactotroph precursors and five types of mature pituitary endocrine cells.GH+PRL+progenitor cells were found transient after birth in the ZBTB20-null mice,and the development of lactotropes was completely blocked due to the complete deletion of GH-PRL+ progenitor cells.At the same time,somatotroph proliferation was inhibited and the cell number decreased.It was found that ZBTB20 deletion itself did not affect the expression of PIT1 in GH+PRL+progenitor cells.ZBTB20 could bind to Prl gene promoter and play a transcriptional activation role on Prl gene.Subsequently,foreign laboratory studies found that ZBTB20 promoted Prl gene expression through the interaction between lncRNA-MIR205HG and PIT1.However,the study cannot fully confirm whether ZBTB20 regulating the lactotropes specification through independent mechanisms and regulating their fate determination after the development occurs and normal physiological function of mature lactotropes or not,because of the limitation of ZBTB20-null mice model.In order to solve the above scientific problems,ZBTB20 Inducible PRL-directed Knockout mice(ZB20-IPO)was established in this study,and the results are as follows.1.Generation of PRL-CreER transgenic mice by CRISPR/Cas9.To analyze the physiological functions of ZBTB20 in lactotropes at different developmental stages,we constructed a lactotrope-specific,Tamoxifen-induced Cre transgenic mouse model.Using CRISPR/Cas9 gene editing technology,the encoding sequence of fusion protein CreERT2 of estrogen receptor(ER)mutant ERT2 and Cre recombinase was introduced into the termination codon of exon 5 of Prl gene in C57BL/6N mice.Through the P2A virus short peptide link sequence(linker)and PRL coding sequence to maintain the same reading frame,play the role of "simultaneous transcription,simultaneous translation".In lactotropes of obtained Prl-CreER offspring mice,the edited Prl allele transcribed mRNA encoding Prl-P2A-CreERT2 under the control of endogenous Prl gene transcription regulatory elements,and simultaneously translated PRL and CreERT2 proteins.CreERT2 binds to Tamoxifen,the specific ligand of ERT2,and enters the nucleus,thus performing the function of Cre-mediated LoxP recombination.To analyze the Cre activity and tissue specificity of Prl-CreER transgenic mice which we mated with Rosa26-tdTomato fluorescence reporter gene mice,and no obvious tdTomato fluorescence signal was observed in the pituitary gland of the obtained heterozygous mice before Tamoxifen induction.The adult heterozygous mice were intraperitoneal injected with Tamoxifen and killed 10 days after induction.Specific tdTomato red fluorescence signal was observed in the pituitary,but no red fluorescence signal was observed in other organs.Further immunohistochemic staining of PRL showed that tdTomato was expressed in most of the pituitary lactotropes,which indicated that the Prl-CreER transgenic mice established could effectively achieve spatio-temporal restriction gene targeting in pituitary lactotropes.2.Regulation of the differentiation of lactotropes by ZBTB20 in developing mice.Suckling mice at different stages of growth and development after birth were selected to be given Tamoxifen administration through breast milk,intraperitoneal injection and intragastric administration.The best induction scheme was determined by considering the tolerance of mother mice and pups as well as the stability and repeatability of induction effect.The pups were given Tamoxifen intragastric administration(75mg/kg)on the 15th day after birth,once a day for 5 days.On the second day after induction,tdTomato fluorescence signal was observed in 70%PRL positive cells of Rosa-tdTomato;Prl-CreER mice.After induction,quantitative RT-PCR results showed that Zbtb20 mRNA level in pituitary gland of ZB20-IPO pups was not significantly different from that of control mice,but Prl mRNA level was decreased by 84%.Immunohistochemical double standard results of anti-PRL antibody and anti-ZBTB20 antibody showed that about 25%PRL positive cells in pituitary of ZB20-IPO mice were negative for ZBTB20,while almost all PRL cells in control mice expressed ZBTB20,indicating that conditional induced knockout of ZBTB20 was successful.Compared with control mice,there was no significant difference in the number of PRL positive cells in pituitary gland of ZB20-IPO pups 21 days after birth.Consistent with this,there was no significant difference in the mRNA level of Drd2,a marker of lactotropes,in the pituitary gland.The 2-hour EdU incorporation labeled showed that the EdU incorporation rate of PRL-positive cells in ZB20-IPO pituitary was 49%lower than that of control mice,and the EdU incorporation rate of PRL+ZBTB20-cells was 57%lower than that of PRL+ZBTB20+ cells.These results indicated that ZBTB20 played an important role in lactotrope proliferation during growth and development.In order to explore the regulation of ZBTB20 on lactotrope progenitor differentiation,Rosa-tdTomato mice were used to trace lactotrope progenitor cells of young mice.Under the above Tamoxifen-induced conditions,the efficiency of PRL positive cells in the ZB20-IPO pituitary gland labeled with tdTomato fluorescence(tdT+)was significantly higher than that of the control pituitary at 21 days after birth,accounting for 88%and 70%of PRL positive cells,respectively.Interestingly,a group of tdT+PRL-cells were found in the pituitary of the control infant mice,accounting for 7.11%of the total tdT+ cells,which was speculated to be derived from the dedifferentiation of lactotropes during development.In the ZB20-IPO pituitary,this group of tdT+PRL-cells increased to 11.8%of the total number of tdT+cells.Considering the close relationship between lactotropes and somatotropes during development,we speculated tdT+PRL-cells have the possibility of differentiating into GH cells.Therefore,we analyzed the expression of GH in tdT+PRL-cells and found that the number of tdT+PRL-GH+cells in ZB20-IPO pituitaries increased to twice of that in the controls,suggesting that ZBTB20 disruption promtes the differentiation of PRL+cells into somatotropes.Immunolabeling of other pituitary hormones in tdT+PRL-cells showed a group of tdT+PRL-ACTH+ cells,which accounted for～0.7%of the total number of tdT+cells,and there was no significant difference between the two groups.We also found a few PRL+ACTH+cells and rare PRL+TSH+ and PRL+FSH+cells in both control and ZB20-IPO pituitaries.These results indicated that the dedifferentiation of lactotropes occurred during differentiation and development,and a small amount of these dedifferentiated lactotropes became somatotropes or corticotropes.ZBTB20 inhibited the dedifferentiation of lactotropes and prevented PRL+precusors differentiating into somatotropes.3.Regulation of lactotrope function by ZBTB20 in adult mice.Adult ZB20-IPO and control mice of the same age and sex were intraperitoneally injected with Tamoxifen(2mg)for 3 times.After 2 days of induction,70%to 80%of PRL+cells in the knockout mice pituitary gland were negative for ZBTB20,while almost all PRL+cells in the control mice pituitary gland expressed ZBTB20.The results showed that ZBTB20 protein expressed by mature lactotropes could be completely degraded within 2 days.Since PRL proteins expressed in mature lactotropes are mostly stored in cells in the form of vesicles and released intermittently in a pulsed manner,we speculated that PRL generated before Tamoxifen induction may remain in cells for some periods of time.Therefore,plasma PRL levels of mice after induction were dynamically observed.The results showed that plasma PRL level of male ZB20-IPO mice was significantly lower than that of control mice from 10 weeks after induction.12 weeks after induction,compared with the control group,the mRNA levels of Zbtb20 and Prl in the pituitary gland of male ZB20IPO mice were significantly decreased.Western blot showed that the protein levels of PRL in the pituitary gland were significantly decreased,but DRD2 mRNA and protein levels were not significantly changed.Immunohistochemical staining showed a normal number of lactotropes in the pituitary gland.The plasma PRL level of female ZB20-IPO mice was significantly reduced after Tamoxifen-induced,resulting in mammary gland hyperplasia disorder.After mating with wild-type male mice,there was no significant difference in the success rate of pregnancy and the number of pups born in the first pregnancy,but the lactation disorder after delivery.These results suggest that ZBTB20 plays an important role in regulating Prl gene expression in mature prolactin cells.In summary,we draw the following conclusions:(1)Using the established PRL-CreER transgenic mice,we have successfully established the inducible prolactin-directed knockout mouse model of ZBTB20.(2)ZBTB20 is not only necessary for the cell fate specification of lactotrope precursors,but also plays an important role in the proliferation and differentiation of lactotropes during postnatal growth and development,partly through inhibiting the dedifferentiation of developing lactotropes.(3)In mature lactotropes of adult mice,as a transcriptional activator of Prl gene,ZBTB20 is essential to maintain the expression and secretion of PRL.In conclusion,this study has clarified the unrecogized function of ZBTB20 in regulating the differentiation and functional homeostasis of lactotropes in the pitutary.