| Objective:Msx2 a homeodomain transcription factor,which encode a protein with 233amino acid and it can suppress the transcription through protein-protein interaction.The research about Msx2 were focused on how Msx2 regulate skull?mammary gland et al development.But recently it was found that Msx2 has essential function in embryonic development,but its role in individual tissue of eye development has not been fully characterized.In lens lineage,Msx2 expression is activated after lens placode induction,and as the lens develops,the expression is localized in the lens epithelium and bow region where mature lens fibers reside.The persistence of the lens stalk resembles a defect in Msx2 mutant mice,and Msx2 defective lenses lose Foxe3 expression,placing Foxe3downstream of Msx2.Lens is the refraction substance of eye ball and it had refraction and accommodation function.It has been proofed that there were lots of ocular tissue development disorder if lens was removed prenatal.Lens was important for eye development.More recently,molecular genetic techniques have been applied to an analysis of lens induction.This has led to the identification of signaling pathways required for lens induction and early lens development.These include the bone morphogenetic protein?Bmp?signaling pathways where Bmp4 and Bmp7 have been implicated.A series of transcription factors involved in early lens development have also been identified.These include Pax6,the Meis transcription factors,Six3,Mab21l1,FoxE3,Prox1 and Sox2.Importantly,analysis has indicated how these elements of the lens induction pathway are related and has defined genetic models to describe the process.Based on the absence of FoxE3 expression in mice carrying a deletion of the Pax6ectoderm enhancer FoxE3 is downstream of Pax6 placode.The similar phenotypes of the dysgenetic lens?FoxE3?and Pax6?EE/?EE mutant mice argue that FoxE3 is upstream of these cellular responses.Similarly,the loss of FoxE3 expression in Mab21l1 mutant mice and the loss of Mab21l1 expression in Pax6 Sey/Sey embryos suggest the Pax6-Mab21l1-FoxE3 gene order indicated.The homeodomain transcription factor Six3 lies genetically downstream of Pax6 placode as mice that do not express placodal Pax6 also do not express placodal Six3.This is also true for Prox1.Since FoxE3 dyl/dyl mice show an expansion of the Prox1 expression domain in the lens epithelium,this suggests that FoxE3 normally suppresses Prox1 at later stages of lens development.In Pax6 Sey/Sey embryos,Sox2expression is not up-regulated in the lens placode suggesting that the late phase of Sox2expression is dependent on Pax6.Similarly,since Sox2?but not Pax6?expression is not up-regulated in the lens placode of Bmp4 null mice,Bmp4 signaling likely contributes to the pathway between Sox2 early and Sox2 late.The lens is derived from the head surface ectoderm of the vertebrate embryo.The first morphological sign of lens development is the formation of the lens placode.This structure is a thickened region of the head surface ectoderm immediately adjacent to the optic vesicle.The thickening of the lens placode occurs only after the optic vesicle has evaginated from the forebrain and made close contact with the surface ectoderm.The interaction between the optic vesicle and presumptive lens ectoderm is extremely strong and mediated by cytoplasmic extensions between the two tissue layers.Subsequently there is a coordinated invagination of the lens placode and outer layer of the optic vesicle.This results in the formation of the lens pit and the optic cup.At this stage,the epithelium of the lens pit closest to the presumptive retina has begun to thicken in the first steps of lens fiber cell differentiation.In addition,the outer layer of the optic cup has folded back against the proximal layer of the optic vesicle to form the adjacent epithelia of the RPE and presumptive retina.Lens pit Closure at the surface ectoderm result in formation of the lens vesicle.Thickening of the posterior epithelium of the lens vesicle continues as fiber cells differentiate and extend towards the lens epithelium.By now the mechanism of how Msx2 regulate ocular development becomes the highlight of research about lens development and some congenital anterior segment disorder disease.As a new member of development regulator,its effection was not clear now.This research mainly focused on how Msx2 regulate lens development and what's the variation of lens cell in Msx2 knockout mice in order to figure out the function of Msx2 in lens development of early stage.Methods:Histological analysisEmbryos or embryonal heads between embryonic day E9.5 to E12.5 and postnatal day?P21?eye balls were fixed overnight in Davidson's solution.Samples were then washed with 70%ethanol and dehydrated through graded ethanol,cleared in xylene,and embedded in paraffin.Sections?5?m?were collected on TESPA-treated slides and stained with hematoxylin and eosin.ImmunohistochemistryEmbryos were fixed for 4 hours in 4%PFA.Samples were then washed with 70%ethanol and dehydrated through graded ethanol,cleared in xylene,and embedded in paraffin.Sections?5?m?were collected on TESPA-treated slides.Antigen retrieval treatment was performed by boiling sections in 0.1 M sodium citrate?pH 6.0?for 20 minutes before adding blocking reagents.Immunostaining was performed using following antibodies:polyclonal rabbit anti-Sox2?1:200 Sigma?,monoclonal anti-Pax6?1:10?,monoclonal anti-Ap-2alpha?1:10?,monoclonal anti-Prox1?1:100 Covance?,and polyclonal rabbit anti-beta crystalline?1:500?.After the addition of primary antibodies,sections were incubated in a humidified chamber overnight at 4°C.Preimmune serum or IgG was used as the negative control.After three washes in PBS,the sections were successively treated with fluorescence labeled secondary antibodies?DyLight TM 549-conjugated Donkey Anti-Mouse Ig G and DyLight TM 488-conjugated Donkey Anti-Rabbit Ig G,Jackson ImmunoResearch Laboratories,Inc?,mounted in fluorescent mounting medium and viewed under a fluorescence microscope?Carl Zeiss Meditec,Thornwood,NY?,and images were captured with a charge-coupled device camera?Spot;Diagnostic Instruments,Sterling Heights,MI?.Labeling of Apoptotic CellsApoptotic cells were detected by terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling?TUNEL?,as described[1]by using an apoptosis detection kit?Fluorescein In situ Cell Death Detection Kit;Roche Diagnostics,Indianapolis,IN?.Briefly,tissue sections were treated with proteinase K.Fragmented DNA was labeled with fluorescein-dUTP,using terminal transferase.Labeled cells were visualized with a fluorescence microscope?Carl Zeiss Meditec,Thornwood,NY?,and images were captured with a charge-coupled device camera?Spot;Diagnostic Instruments,Sterling Heights,MI?.BrdU Labeling of Proliferating CellsTimed-pregnant mice were injected intraperitoneally with 100 mg of BrdU per gram of body weight.One hour later,animals were sacrificed.Embryos were dissected and fixed in Methacarn?Methanol:chloroform:Glacial acetic acid=6:3:1?overnight?at 4°C?.Embryos were then dehydrated through graded ethanol and embedded in paraffin.Sections were cut?5?m?,deparaffinized,and soaked in 3%hydrogen peroxide in methanol for 10minutes.They were washed three times in PBS.Subsequently,to depurinate DNA,sections were incubated in freshly prepared 2N HCl for 20 minutes at room temperature and neutralized in 0.1M sodium borate?pH 8.5?for 10 minutes.Sections were then rinsed three times in PBS.Immunodetection of BrdU was performed using the 5-Bromo-2'-deoxy-uridine Labeling and Detection Kit II?Roche?,according to the manufacturer's instructions.Whole-mount in situ hybridizationMsx2cko mice were time mated,and the day of the vaginal plug was defined as embryonic day 0.Tissues for in situ hybridization were fixed by immersion in 4%paraformaldehyde in phosphate-buffered saline?PBS?overnight.In situ hybridization was performed as previously described by Jensen and Wallace?1997?.The following templates were used to generate digoxigenin-labelled antisense RNA probes,as previously described:murine FoxE3,was transcribed using T7 RNA polymerase from a NotI linearized template;murine Sox2,was transcribed using T3 polymerase from an BglII linearized template.MicroarrayCytoscape software was used for differentially expressed gene?DEGs?molecular function ClueGO analysis.DEGs were screened using SAS?SBC Analysis System?statistical software online?http://sas.ebioservice.com/?.To identify the functions of DEGs,gene ontology?GO?and KEGG pathway enrichment analysis were carried out using SAS software online.Real-time PCRRNA specimens were extracted from Msx2CKO and Msx2flox/flox lens of three independent litters respectively at post-natal day?P?1 using RNeasy micro kit?Cat#74004,QIAGEN,GmBH,Germany?.qPCR was carried out to verify the microarrays results using SYBR Premix Ex TaqTM II?Takara,Dalian,China?and analyzed based on the equation RQ=2-??CT.The procedure was 94?for 2min,followed by 40 cycles at 94?for 1min and at60?for 40s.Primers:Casp8?forward 5'-ccacgagattctagaaggctaccaaagcgc-3',reverse5'-ctcacgtcatagttcacgccagtca-3'?;Casp3?forward 5'-ccatggtgaaggggtcatttatgggaca-3',reverse5'-tggacacaatacacgggatctgtttctttg-3'?.Result:Cre/IoxP-mediated selective deletion of Msx2We generated the floxed Msx2 mouse line with Cre/LoxP-mediated selective deletion of Msx2.By E10.5,whole-mount in situ hybridization of the generated Msx2cko lens failed to detect any signal that means Msx2 also lose function at the same time.There are big differences between P21 Msx2cko and wild-type mice by gross inspection.The Msx2cko mice had no eyelashes and lacked hair on the surface of the eye lids and in a stripe that ran from the temporal to the nasal side of the eye.Histological sections of these P21 eye show reduction in the size of lens,the nuclei of the lens fibers are displaced toward the anterior and posterior of the lens and the cortical fiber cells are vacuolated.Early lens development in Msx2cko miceWe examined early lens development in Msx2cko mice.At E9.5,when the optic vesicle had close contacted the lens placode,no structural difference was observed between the developing eyes of Msx2cko mice and that of wild-type littermates.In contrast at E 10.5,the lens vesicle that had invaginated into the optic cupwas considerably large in the developing eyes of wild-type mice than in those of Msx2cko mice.At E 11.5,the lens vesicle had closed in the eyes of wild-type mice,while the lens vesicle was also closed but appeared small size.Subsequently at E 12.5,the epithelium of the lens vesicle had completely separated from the surface ectoderm in wild-type mice,while it was still continuous adherent with it in Msx2cko mice.In summary,the examination of early eye development in Msx2cko mice strongly indicates that the small lens size and persistence of the lens stalk are associated with a substantial delay in lens formation.Increasing apoptosis contributes to the delay in lens formationSimilar to previous gene mutation mice,the reduction in lens size has also been observed in Msx2cko mice.To assess if changes in mitotic and apoptosis activity are involved in the delay of lens formation in Msx2cko mice,embryos and BrdU injected embryos were examined by immunohistochemistry.At E10.5,even still no obvious difference was observed on BrdU-positive cells between Msx2cko mice and wild-type embryos,but the considerably more apoptosis cells were observed in lens placode and optic cup during the invagination in Msx2cko mice when compared with their wild-type littermates.Caspase-3 and Caspase-8 were increased in Msx2cko lens at P1.Based on both BrdU and apoptosis experiments,we think that more activity of apoptosis in lens placod take a major role in delayed lens formation and the development of a small eye in Msx2cko mice.The expression of lens specific transcript factor in Msx2cko miceTo understand the interactions among the Msx2,Pax6,Sox2,Foxe3,Prox1 and AP-2?,we monitored the expression of Pax6,Sox2,Foxe,Prox1 and AP-2?in Msx2cko mice by performing immunohistochemistry.Immunostaining results show the expression patterns of Pax6,Sox2 and AP-2?are same as earlier findings in the developing mouse eye.The distribution and intensity of the staining in Msx2cko mice embryos were not different from that in wild-type littermates.Our results indicated that the expression of Pax6,Sox2 and AP-2?is not affected by Msx2 conditional deletion.In contrast to Pax6,Sox2 and AP-2?,we investigated FoxE3 expression by whole-mount in-situ hybridization.We observed at E10.5 the FoxE3 expression level was significantly reduced during lens development in Msx2cko mice embryos compared with their wide-type littermates.Conclusion:1.There's lens development disorders in Msx2cko mice and its phenotype are small lens.Lens and cornea adhere and detach retardate.2.At E10.5,no obvious difference was observed on BrdU-positive cells between Msx2cko mice and wild-type embryos,more apoptosis cells were observed in lens placode and optic cup during the invagination in Msx2cko mice when compared with their wild-type littermates.Casp3 and Casp8 were ugregulated in Msx2cko mice at P1.3.We observed the FoxE3 expression level was significantly reduced during lens development in Msx2cko mice embryos compared with their wide-type littermates by whole-mount in-situ hybridization... |
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