Tsc1 Gene Regulatory Mechanism Study Vascular Development In The Mouse Lung Development

Rationale:Tuberous sclerosis comples (TSC) is an autosomal-dominant genetic disorder, resulted from mutations in TSC1or TSC2genes. Mutations in either tuberous sclerosis complex (TSC), Tscl or Tsc2abrogate the suppression of TSC1/TSC2protein complex on the signaling controlled by mechanistic target of rapamycin (mTOR). The consequent hyperactivation of mTOR is the major mechanism of TSC disease, which features multiple aberrant proliferation lesions, including epilepsy, mental retardation, skin angiofibroma, fetal cardiac rhabdomyomsas, pulmonary lymphangioleiomyomatosis, renal angiomyolipoma, and liver hemangiomas. Therefore, the significance of TSC1/2-mTOR signaling pathway in angiogenesis should be determined.Objective:To examine the physiological role of the TSCl/2-mTOR signaling pathway in angiogenesis and embryogenesis, and its clinical relevance for TSC progression, a Cre-lox conditional gene knockout approach was used to inactivate Tscl in mouse vascular endothelial cells.Methods and Results:Overactivation of mTOR signaling was present in the endothelia of Tscl mutant mice. This mouse model was embryonic lethal mostly at E14.5due to severe phenotype defects in embryos and yolk sacs, including subcutaneous edema, hemorrhage and angiogenesis defects. Whole-mount embryo immunostaining revealed poor-structured hierarchic organization of vessels, little evidence of intricate network of vessels, irregularly shaped blood vessels and decreased number of distal vessels were observed in the KO embryos. In addition, poorly developed trabeculae and a thinner ventricular wall in the heart. It was disrupted the balance of proliferation and apoptosis in endothelia of mutant embryos, which resulted in their increase apoptosis. Enlarged endoplasmic reticulum was observed in abnormal endothelial cells. Compared to littermate controls, mutant embryos showed abnormal expression of angiogenic factors. Prenatal rapamycin treatment at E12to E13days rescued the angiogenesis defects, embryonic lethality and prolonged (extended) the survival of the mutant mice up to22days after birth. Although partial the mutant mice have no obvious phenotypes at birth, it had very poor weight gain and hypogenesis which led to death after birth, in comparison to their control littermates.Conclusions:Endothelial cell expression of Tscl and physiological mTOR signaling are crucial for vascular development and embryogenesis. Disruption of normal angiogenic pathways by hyperactive mTOR signaling may contribute to TSC pathogenesis. The study of vascularized disorder of TSC contributed to better understand the pathogenesis of pulmonary lymphangioleiomyomatosis and renal angiomyolipoma. Alveolar epithelial type Ⅱ (AECⅡ s) cells are crucial for pulmonary physiology and pathophysiology, served as the progenitor of alveolar epithelial type II cells and lung defender. Despite dysfunction of AEC Ⅱ s involved in several lung disorders, the role in targeted genes remained undefined. It is required for established transgenic mice models of respiratory epithelium. Several previous studies have described inducible Cre expression systems (SPC-rtTA/TetO-Cre, SPC-Cre-ERT2) were applied to investigate underlying mechanisms of targeted genes in AEC II s. However, some limitations were revealed to hamper the study of specific genes function in these systems. Non-inducible Cre expression system (SPC-Cre) is the better powerful tool to insight into gene function during the processes of both normal development and disease. SPC-Cre line was applied in previous study, but more details about the generation of the mouse models haven’t been illustrated. Others, It was reported that SPC-Cre showed abnormal dilated cysts. Here we reported a non-inducible Cre mouse line in which conditional system controlled by SPC gene promoter to express Cre recombinase was used and generated a transgenic mouse SPC-Cre line, based on C57BL/6J background. In order to test the specificity of Cre recombinase in AEC II s, SPC-Cre mice were crossed with ROSA26R reporter and Tsclfx/fx mice, respectively. X-Gal staining and PCR results suggested β-Galactosidase positive cells located in AEC II s cells from SPC-Cre/ROSA26R mice lung sections and mutant allele was present in lung tissues of SPC-Cre/Tscl-/-.It should be a useful tool to investigate AEC Ⅱs function in lung development and lung disorders. Current results about lung development indicated that SPC-Cre/Tscl-/-presented a normal gross morphology. While histology analysis showed that enlargement of alveolar spaces and numbers of alveolar septum decreased in SPC-Cre/Tscl-/-. Transcript factor levels of lung tissue were affected in mutant mice. More mechanisms of the defects need to be further studed.