The Function Of BMP4 Downstream Signal Molecular Smad1 In Lung Development

Lung development is a complicate process controlled by cooperation of growth factors.Branching morphogenesis is the main process during early lung development stage. Primary airway tube continuous growing, repeated branching to form a respiration tree. After that the terminals of airway expend and form terminal sac structure and develop into alveoli at last. Lung airway epithelium tissue differentiate into type I and type II epithelium cell which can secret surfactant at the same time and at last form a mature air - exchange surface by tightly combine between epithelium and beneath pulmonary capillaries bed. Whether the premature baby can live or not depend on the mature extent of embryo lung. On the other hand, COPD ( Chronic Obstructive Pulmonary Disease ) , characterized by a gradual lose of lung function, is one of the main diseases give serious bad effect on the health and life quality of the elderly. COPD is the fourth cause of death in United States. According to the statistic data from China, respiratory system diseases are the third cause of death in China city resident in 2003. More than 85% of cases are chronic lower respiratory tract disease that is similar to COPD. Therefore, it is important to deeply understand of the molecular mechanism of lung morphogenesis and function establishment to find new method to promote lung maturation of premature lung and repair damaged adult lung.BMP4 ( Bone morphogenetic protein 4 ) is a key growth factor that plays an important role in the establishment of axis information and the formation of many organs during embryo development. Smad1, Smad5 and Smad8 are intracellular signaling molecule that can transmit cell surface BMP4 signal to nucleus to control target gene expression. BMP4 express in the distal lung tissues and control proximal - distal differentiation of embryo lung tissue during mouse lung devel -opment. Peripheral lung tissue would be dysplasia if BMP4 pathway is inhibited in embryo lung. The mechanism of how BMP4control lung development is still unknown.This research project is to analyze the function of Smadl, a BMP4 pathway downstream signal molecular, in lung development. The expression pattern of smadl in different development stage lung was checked and the function of smadl in lung branching morphogenesis control was analyzed in mouse embryo lung organ culture system. There are start code and translation control sequence in the exon 2 of smadl gene. The function of epithelium smadl in peripheral lung development was analyzed in transgenic mouse model that can produce smadl null mutant by specifically knockout exon 2 of mouse smadl gene. No smadl protein can be translated in lung epithelium.Material and Method1. Expression pattern of smadl during lung development. Collect E12.5, E14.5 , E16.5 and E18.5 mouse embryo from timed - pregnant Swiss - Webster mouse. Dissect mouse embryo, collect lung. Some lung samples are fixed with 4% paraformaldehyde, then dehydrate embedding and cut section. Check smadl expression pattern in different development stage embryo lung by immu-nochemistry. To determine the type of smadl positive cell, Smadl and a -smooth muscle actin (a -SMA) , a marker for smooth muscle cells and myofi-broblast cells, were localized by co - immunofluorescence staining. Other lung samples are frozen immediately, then lysed with RIPA buffer. Smadl protein expression level was determined by Western - ECL.2. Smadl function in lung branching morphogenesis. Dissect and collect Ell. 5 embryo lung from timed - pregnant Swiss - Webster mouse. Embryo lungs were cultured in BGJb medium. Medium was changed every two days. Smadl antisense oligonucleotide was added to medium at a final concentration of 40|xM. Sense oligonucleotide, with complementary sequence of antisense oligonucleotide, was added to the same concentration as a control group. Set up another normal organ culture group. Ten embryo lungs cultured in each group. Af-ter four days, collect lung explants, take picture and count the number of epithelium sac visible around periphery of the lung explants as a standard to evaluate branching morphogenesis. Student's t - test was used for statistic analysis. Fix, embed and cut lung explants section. Check PCNA index by immunostain. Lyse lung explants to purify total RNA. Real - time PCR check the expression level of peripheral lung differentiation marker SP - C and proximal lung differentiation marker CC10.3. How smadl null mutant in embryo lung epithelium influence lung development. Three C57BL background transgenic mouse lines contain SP - C -rtTA~/t*^(tet0)1 - CMV - Cre~/tgand Smadllo"'/Uap were used to generate triple transgenic male mouse with SP - C - rtTA~/tg/ {tetO)l - CMV - Cre*"*/ Smadl */Uap . Then this male triple transgenic mouse crossed with female SP - C - rtTA-'~/ (tetO)l - CMV - Cre~/~/Smadlloxp/loxl'lransgemc mouse. Doxycyc-line was added to food and water from E8.5 to the experiment date. Collect specific development stage mouse embryo lungs. Fix, cut section, and HE stain. Purify total RNA. Analyze the expression level of lung tissue differentiation marker and other molecule that can control lung development by Real - time PCR or immunostain. Affymetrix Mouse Genome 430 2.0 Array was used to analyze whole genome expression level change in El8. 5 transgenic mouse lung.Experiment Result1. Smadl express pattern during mouse lung development;Smadl expressed in peripheral lung tissue during lung development. The majority Smadl protein was detected in the peripheral airway epithelium in El2. 5. Only a few mesenchyme cells expressed Smadl in this stage. Compared with E12. 5, airway epithelium Smadl expression level reduced in El4. 5. Majority Smadl protein was localized in mesenchyme in El6. 5 and El8. 5. Smadl expression level is higher in early embryo lung development stage (E12. 5 and E14. 5) than that in later embryo lung development stage (E16.5 and E18.5). Some Smadl protein was co - localized with a - SMA in smooth muscle cell around large airway and blood vessels and some mesenchyme myofibroblast cell in El6. 5.2. Smadl function in lung branching morphogenesis. Smadl antisense oli-gonucleotide reduced smadl expression level in lung explants. At the same time branching morphogenesis was significantly inhibited and terminal airway branches number was reduced 20%. The PCNA immunostain result showed the inhibition of lung explants epithelium cell proliferation. Proximal and distal lung differentiation marker CC10 and SP - C expression level was significantly reduced. Overexpression Smadl in lung airway epithelium cannot promote lung branching morphogenesis.3. The function of smadl expressed in embryo lung epithelium. Smadl is an indispensable factor to participate lung development control in each stage of normal lung development. Completely delete smadl in lung epithelium ( Smadls/A ) , newborn mouse lung was dysplasia, The Smadl^* transgenic mouse shows dyspnoea, body color was violet and died in half an hour after birth. Autopsy result shows that lung cannot open. Newborn Smadl^A mouse still cannot live even Doxycycline induction started in E17.5.Analysis of El8.5 SmadlA/A mouse embryo lung showed that the weight ratio of Lung and body was significantly decreased compared with control group mouse embryo lung. Morphological analysis result of HE stain pictures showed that the airspace area in peripheral lung tissue of SmadlA/A mouse was significantly increased compared with control group mouse embryo lung, which has normal smadl expression. There was peripheral lung structure hypoplasia in SmadlA/A mouse. Perhaps this was result from abnormal branch morphogenesis. Type II epithelium cell marker SP - C can hardly be found in immunostain. Real - time PCR showed significantly decrease of mRNA expression level of type I and II epithelium cell marker such as Aqp5 , SP - C etc. There was peripheral lung epithelium cell hypoplasia. Immunostain result of TTF1 and Foxa2 showed dramatic expression pattern change of these transcription factors. In peripheral lung tissues TTF1 expression level was decreased but Foxa2 expression increased. HE stain result showed proximal airway developed well. Immunostain showed that (3 - tubulin can only be found at proximal airway. (3 - tubulin expression pattern was correct. Microarray result showed that expression level of SP - A, Aqp5 and several other genes changed more than 2 times inmouse lung compared with control group mouse embryo lung. Many of these genes participate immunoreaction control, anti - harmful environment factor or lipid metabolism that relate to surfactant lipid synthesis. Maybe there is dose dependent effect for lung epithelium smadl. TheSmadl*'* newborn mouse, which only exon 2 knocked out from one smadl allele, showed similar phenotype as Smadl*'* mouse, which exon 2 knocked out from two smadl allele. Smadl*'* newborn mouse also show dyspnoea, body color violet but in a less extend and these mice can live 4 to 5 hours after birth.Conclution1. Smadl expresses in peripheral lung tissue in a dynamic expression pattern. In specific lung development stage, smooth muscle cell and myofibroblast cell in lung mesenchyme express smadl. Smadl may play important role in the differentiation of these two type cells.2. Smadl plays important role in embryo lung development. Smadl participates branching morphogenesis control in early embryo lung development stage. It is an indispensable factor that can promote airway branching morphogenesis. In middle and later embryo development stage Smadl plays important role in peripheral lung tissue development, especially in type I and H lung epithelium cell differentiation control.