Study On The Mechanism Of Histone Deacetylase 3 In Mouse Embryo Development

Bronchopulmonary dysplasia (BPD) is a form of lung disease that affects premature newborns. The pathogenesis of this condition remains complex and poorly understood; however various factors can not only injure small airways but also interfere with alveolarization (alveolar septation), leading to alveolar simplification with a reduction in the overall surface area for gas exchange. The developing pulmonary microvasculature can also be injured. The etiology of BPD is poorly understood, the cause of BPD is related to preterm, inflammation life-saving oxygen and mechanical ventilation. Studies show that heredity may play a role in causing BPD. The aim of this study is to examine the roles of epigenetic factors in the BPD progression.Histone deacetylases (HDACs) are a group of important epigenetic factors that mediate gene repression. One important way to induce gene repression by HDACs acts by deacetylating the histones, which compacts the chromatin and reduces access of transcriptional regulators. In addition, HDACs have also been shown to modulate the acetylation status of some transcription factors including p53 and Gata4 to control their transcriptional activities.The mammalian HDAC superfamily consists of 11 members that can be classified into four classes. These four classes of HDACs, class Ⅰ, Ⅱa, Ⅱb and Ⅳ have distinct structures and functions. The class Ⅰ family of HDACs is composed of four members, HDAC1, HDAC2.HDAC3 and HDAC8. A common function of class Ⅰ HDACs revealed by numerous studies is that they play a crucial role in promoting cell proliferation and inhibiting apoptosis. In addition to this common function, recent mouse genetic studies demonstrated that class Ⅰ HDACs play tissue-specific roles in various organ development.Our recent studies have identified the specific roles for different members of class I HDACs in regulating lung epithelial development. Epithelial Hdac1/2 are required for the development and regeneration of Sox2+proximal lung endoderm progenitor cells, whereas epithelial HDAC3 is crucial in regulating AT1 cell spreading and remodeling during lung sacculation. In addition to its epithelial expression, HDAC3 is also highly expressed in the lung mesenchyme throughout embryonic development, suggesting a potential mesenchymal-specific role of HDAC3 in promoting lung development.Part OneLoss of HDAC3 in the mesenchyme results in lung hypoplasiaTo determine the expression pattern of HDAC3 during lung development, we performed the immunohistochemistry for HDAC3 expression at various stages of lung development. From E12.5-E18.5, HDAC3 broadly expressed in the epithelial and mesenchymal compartments. Given the high expression level of HDAC3 in the mesenchyme throughout embryonic lung development, HDAC3 may potentially play a critical role in regulating lung mesenchymal cell development and differentiation. To further investigate the functional roles of HDAC3 in the mesenchyme of developing lungs, we generated a tissue-specific deletion of HDAC3 using the Hdac3flox allele and Dermo1Cre line, which efficiently drives Cre recombination in the mesoderm beginning at approximately E9.5, these Hdac3flox/flox:Dermo1Cre mutants will now be referred to as Hdac3Dermo1creKO.Examination of HDAC3 expression in Hdac3Dermo1creKO lungs by immunohistochemistry showed that HDAC3 protein was efficiently depleted in the lung mesenchyme, but retained its expression in the lung epithelium, in contrast to the control lungs where HDAC3 staining could be found in both epithelium and mesenchyme. All Hdac3Dermo1creKO mice died at birth due to apparent respiratory distress (data not shown). To explore the reason for perinatal lethality in Hdac3Dermo1creKO mutants, histological analysis was performed on embryonic lungs from E13.5 to E18.5. At E15.5, Hdac3Dermo1creKO mutant lungs were slightly smaller than controls. The reduced sizes of mutant lungs were further shown by H&E stainings at E16.5. However, the branching morphogenesis of lung epithelial cells appeared to be normal in the Hdac3Dermo1creKO mutant lungs at this stage. Notably, histological examinations of Hdac3Dermo1creKO lungs at E18.5 revealed a severe lung sacculation defect as evidenced by reduced distal airspace and thickened septa. Taken together, these results suggested that tissue-specific deletion of HDAC3 resulted in lung mesenchymal hypoplasia and sacculation defects during mouse lung development.Part TwoHDAC3 effects mesenchymal cell proliferation and differentiation during lung developmentGiven the smaller size of the Hdac3Dermo1creKO lungs between E15.5 and E18.5, we hypothesized that HDAC3 deficiency in lung mesenchyme may result in decreased cell proliferation or increased apoptosis in either lung mesenchyme or epithelium. To assess whether cell proliferation was affected, we performed phospho-histone H3 (PO4-H3) and BrdU immunohistochemistry on E16.5 and E18.5 lung sections. Less BrdU+TTF-1-cells were observed in Hdac3Dermo1creKO lungs compared to control lungs, indicating a decreased proliferation rate of mesenchymal cells. Moreover, the percentage of BrdU+ cells within total TTF-1+epithelial population was similar between the Hdac3Dermo1creKO mutant and control lungs at E18.5, suggesting that the lung epitheial proliferation rate was unaffected.HDAC3 is a well-known regulator of cell cycle progression. Previous work has demonstrated that HDAC3 play a crucial role in regulating the expression of multiple cell cycle-related genes during development and tissue regeneration. To test the hypothesis that the lung mesenchymal proliferation defects of Hdac3Dermo1creKO mutants involved disturbed cell cycle progression, we used Q-PCR to investigate a panel of cell cycle-related genes and found mRNA levels of cyclinA2, cyclinB1, cylcinD1 and CDK1 significantly reduced, with an increase in cyclin-dependent kinase (CDK) inhibitor P27 in the Hdac3Dermo1creKO lungs, indicating that mesenchymal HDAC3 is dispensable for lung epithelial cell proliferation but is required for maintaining proper mesenchymal proliferation through regulating the expression of cell cycle-related genes.During development, the lung mesenchyme generates multiple cell lineages, including smooth muscle cells (SMCs), endothelial cells, pericytes and interfib rob lasts. To assess whether HDAC3 plays a role in regulating lung mesodermal cell lineages differentiation, we performed immunohistochemistry for cell specific markers.1. Double staining of SM22 and Sox2 shows that airway smooth muscle differentiation and development are not altered in Hdac3Dermo1creKO lungs at E18.5.2. PECAM staining shows that vascular development appears to be normal in Hdac3Dermo1creKO lungs as well. Q-PCR analysis of SM22 and PECAM marker reveal no significant changes in Hdac3Dermo1creKO lungs compared to control lungs.3. Q-PCR data showed a decrease of multiple markers for interstitial fibroblasts including Pdgfr-b and Vimentin in Hdac3Dermo1creKO mutant lungs Immunostaining on Pdgfr-b demonstrated that there was a reduction in the total number of Pdgfr-b+ interstitial fibroblasts rather than attenuated expression level of the protein per cell. Consistent with our in vivo results, the cultured Hdac3Dermo1creKO lung mesenchymal cells showed a significant defect in proliferation as indicated by the decreased total number of Pdgfrb+cells and decreased percentage of Ki67+Pdgfr-b+cells.Part ThreeHDAC3 instructs alveolar type I cell differentiation by regulating a Wnt signaling nicheAt around E17.5 in the mouse, lung development switches from branching morphogenesis to the saccular stage. These processes of lung sacculaion involve expansion of airspace and alveolar epithelial differentiation. AEC1 and AEC2 cells are the major source of distal epithelial cell lineages, their differentiation and maturation was primary events that occur during this stage. To determined whether mesenchymal deletion of HDAC3 affect alveolar epithelial cell differentiation, we performed the Immunostaining and Q-PCR analysis to examined the expression of cell type-specific markers for epithelial cells.1. Immunohistochemistry and quantitative PCR (Q-PCR) results showed that expression of AEC1 cell markers (T1 alpha and Aqp5) in Hdac3Dermo1creKO mutant lungs were greatly attenuated at E18.5.2. Differentiation of alveolar type II cell (AEC2) cells appeared normal as measured by the expression of AEC2 cell markers including Sftpc, Sftpb and Abca3.3. Immunostaining and Q-PCR for proximal epithelial markers, including Scgb1a1 (secretory Clara cells), beta-Tubulin IV and Foxj1 (ciliated epithelial cells), were performed on E18.5 lungs and showed no significant differences in Hdac3Dermo1creKO mutant lungs. Together, these data suggest that mesenchymal HDAC3 is specifically required for AEC1 cell differentiation, but is dispensable for AEC2 cell, ciliated cell and secretory cell differentiation.To identify molecular changes that could potentially lead to the AEC1 cell phenotype, we isolated the mesenchymal/epithelial cells and performed Q-PCR analysis. The Hdac3Dermo1creKO mutant mesenchymal cells showed a significant decrease in both Wnt5a and Wnt2. In addition, we also observed a down-regulation of Wnt5a and Wnt7a expressed by mutant epithelium). To further determine if Wnt/p-catenin signaling activity was impaired in the mutant lung epithelium due to decreased Wnt ligands, we performed epithelial Q-PCR analysis on several well-known canonical Wnt targets including Axin2, Lef-1 and Cyclin D1 and observed a decrease in all these target gene expression in the mutant epithelium. These data indicated that loss of mesenchymal HDAC3 could disrupt the expression level of several Wnt ligands, resulting in a reduction of canonical Wnt activity in the lung epithelium, which could potentially contribute to the impairment in AT1 cell differentiation and lung sacculation. We have shown that mesenchymal HDAC3 is required for late lung development and AT1 cells differentiation. Importantly, the AT1 cell differentiation defects caused by down-regulateion of Wnt/β-catenin signaling expression in lung epithelium can be partially rescued in vivo by using the pharmacological activator LiCl. Therefore, our data may provide novel mechanistic insights on how neonatal respiratory diseases occur and what genes can be potentially targeted for new therapies.