Late gestational lung hypoplasia in a mouse model of the Smith-Lemli-Opitz syndrome

BACKGROUND: Normal post-squalene cholesterol biosynthesis is important for mammalian embryonic development. Neonatal mice lacking functional dehydrocholesterol Δ7-reductase (Dhcr7), a model for the human disease of Smith-Lemli-Opitz syndrome, die within 24 hours of birth. Although they have a number of biochemical and structural abnormalities, one cause of death is from apparent respiratory failure due to developmental pulmonary abnormalities. RESULTS: In this study, we characterized further the role of cholesterol deficiency in lung development of these mice. Significant growth retardation, beginning at E14.5~E16.5, was observed in Dhcr7(-/- )embryos. Normal lobation but smaller lungs with a significant decrease in lung-to-body weight ratio was noted in Dhcr7(-/- )embryos, compared to controls. Lung branching morphogenesis was comparable between Dhcr7(-/- )and controls at early stages, but delayed saccular development was visible in all Dhcr7(-/- )embryos from E17.5 onwards. Impaired pre-alveolar development of varying severity, inhibited cell proliferation, delayed differentiation of type I alveolar epithelial cells (AECs) and delayed vascular development were all evident in knockout lungs. Differentiation of type II AECs was apparently normal as judged by surfactant protein (SP) mRNAs and SP-C immunostaining. A significant amount of cholesterol was detectable in knockout lungs, implicating some maternal transfer of cholesterol. No significant differences of the spatial-temporal localization of sonic hedgehog (Shh) or its downstream targets by immunohistochemistry were detected between knockout and wild-type lungs and Shh autoprocessing occurred normally in tissues from Dhcr7(-/- )embryos. CONCLUSION: Our data indicated that cholesterol deficiency caused by Dhcr7 null was associated with a distinct lung saccular hypoplasia, characterized by failure to terminally differentiate alveolar sacs, a delayed differentiation of type I AECs and an immature vascular network at late gestational stages. The molecular mechanism of impaired lung development associated with sterol deficiency by Dhcr7 loss is still unknown, but these results do not support the involvement of dysregulated Shh-Patched-Gli pathway in causing this defect

The Epicardium and the Development of the Atrioventricular Junction in the Murine Heart

Insight into the role of the epicardium in cardiac development and regeneration has significantly improved over the past ten years. This is mainly due to the increasing availability of new mouse models for the study of the epicardial lineage. Here we focus on the growing understanding of the significance of the epicardium and epicardially-derived cells in the formation of the atrioventricular (AV) junction. First, through the process of epicardial epithelial-to-mesenchymal transformation (epiEMT), the subepicardial AV mesenchyme is formed. Subsequently, the AV-epicardium and epicardially-derived cells (EPDCs) form the annulus fibrosus, a structure important for the electrical separation of atrial and ventricular myocardium. Finally, the AV-EPDCs preferentially migrate into the parietal AV valve leaflets, largely replacing the endocardially-derived cell population. In this review, we provide an overview of what is currently known about the regulation of the events involved in this proces

Mef2c regulates transcription of the extracellular matrix protein cartilage link protein 1 in the developing murine heart.

Cartilage Link Protein 1 (Crtl1) is an extracellular matrix (ECM) protein that stabilizes the interaction between hyaluronan and versican and is expressed in endocardial and endocardially-derived cells in the developing heart, including cells in the atrioventricular (AV) and outflow tract (OFT) cushions. Previous investigations into the transcriptional regulation of the Crtl1 gene have shown that Sox9 regulates Crtl1 expression in both cartilage and the AV valves. The cardiac transcription factor Mef2c is involved in the regulation of gene expression in cardiac and skeletal muscle cell lineages. In this study we have investigated the potential role of Mef2c in the regulation of ECM production in the endocardial and mesenchymal cell lineages of the developing heart. We demonstrate that the Crtl1 5' flanking region contains two highly conserved Mef2 binding sites and that Mef2c is able to bind to these sites in vivo during cardiovascular development. Additionally, we show that Crtl1 transcription is dependent on Mef2c expression in fetal mitral valve interstitial cells (VICs). Combined, these findings highlight a new role for Mef2c in cardiac development and the regulation of cardiac extracellular matrix protein expression

Wnt/β-catenin and sonic hedgehog pathways interact in the regulation of the development of the dorsal mesenchymal protrusion

The dorsal mesenchymal protrusion (DMP) is a second heart field (SHF) derived tissue involved in cardiac septation. Molecular mechanisms controlling SHF/DMP development include the Bone Morphogenetic Protein and Wnt/β-catenin signaling pathways. Reduced expression of components in these pathways leads to inhibition of proliferation of the SHF/DMP precursor population and failure of the DMP to develop. While the Sonic Hedgehog (Shh) pathway has also been demonstrated to be critically important for SHF/DMP development and atrioventricular septation, its role in the regulation of SHF proliferation is contentious. Tissue-specific deletion of the Shh receptor Smoothened from the SHF resulted in compromised DMP formation and atrioventricular septal defects (AVSDs). Immunohistochemical analysis at critical stages of DMP development showed significant proliferation defect as well as reduction in levels of the Wnt/β-catenin pathway-intermediates β-catenin, Lef1, and Axin2. To determine whether the defects seen in the conditional Smoothened knock-out mouse could be attributed to reduced Wnt/β-catenin signaling, LiCl, a pharmacological activator of this Wnt/β-catenin pathway, was administered. This resulted in restoration of proliferation and partial rescue of the AVSD phenotype. The data presented suggest that the Wnt/β-catenin pathway interact with the Shh pathway in the regulation of SHF/DMP-precursor proliferation and, hence, the development of the DM

Expression of the BMP receptor Alk3 in the second heart field is essential for development of the dorsal mesenchymal protrusion and atrioventricular septation

The dorsal mesenchymal protrusion (DMP) is a prong of mesenchyme derived from the second heart field (SHF) located at the venous pole of the developing heart. Recent studies have shown that perturbation of its development is associated with the pathogenesis of atrioventricular (AV) septal defect. Although the importance of the DMP to AV septation is now established, the molecular and cellular mechanisms underlying its development are far from fully understood. Prior studies have demonstrated that bone morphogenetic protein (BMP) signaling is essential for proper formation of the AV endocardial cushions and the cardiac outflow tract. A role for BMP signaling in regulation of DMP development remained to be elucidated. To determine the role of BMP signaling in DMP development. Conditional deletion of the BMP receptor Alk3 from venous pole SHF cells leads to impaired formation of the DMP and a completely penetrant phenotype of ostium primum defect, a hallmark feature of AV septal defects. Analysis of mutants revealed decreased proliferative index of SHF cells and, consequently, reduced number of SHF cells at the cardiac venous pole. In contrast, volume and expression of markers associated with proliferation and active BMP/transforming growth factor β signaling were not significantly altered in the AV cushions of SHF-Alk3 mutants. BMP signaling is required for expansion of the SHF-derived DMP progenitor population at the cardiac venous pole. Perturbation of Alk3-mediated BMP signaling from the SHF results in impaired development of the DMP and ostium primum defect

Sequence alignment of the mouse, rat, and human Crtl1 (Hapln1) promoters.

<p>The mouse, rat, and human Crtl1 genes, plus 1000 bp of the upstream promoter, were aligned using the web-based tool Kalign. Using this alignment, two conserved Mef2 consensus sites were identified at positions −698 to −707 and −913 to −923.</p

Crtl1 and Mef2c expression in the AV junction at ED14.5 and ED17.5.

<p>(A) Atrioventricular junction in an H&E stained ED14.5 specimen. (B) Immunofluorescent staining of Crtl1 (green) shows Crtl1 is expressed in the ventricular aspect of the leaflets of the mitral valve at ED14.5, Mef2c (red, panel B) is also expressed throughout the leaflets of the mitral valve. (C) Atrioventricular junction in an H&E stained ED17.5 specimen. (D) Crtl1 is expressed sub-endocardially on the atrial aspects of the mitral valve leaflets at ED17.5 and Mef2c (red, panel F) is expressed in both the mesenchyme and endocardial lining of the leaflets, colocalizing with Crtl1 protein expression (green, panels D).</p

Mef2c binds to the Crtl1 Promoter in the developing heart.

<p>(A) Immunoblot for Mef2c protein. Lane 1, Mef2c binding to control Mef2 consensus sites from the muscle creatine kinase gene. Lane 2, negative control oligo with mutated Mef2 sites fails to bind to Mef2c. Lanes 3 and 5, Mef2c interacts with the Mef2 consensus sites in the Crtl1 promoter region at −913 to −923 (Site 1, Lane 3) and at −698 to −707 (Site 2, Lane 5). Lanes 4 and 6, mutation of the Mef2 consensus sites in the Crtl1 promoter region blocks Mef2 binding at both consensus sites. (B) Mef2c and Sox9 bind the Crtl1 Promoter <i>in </i><i>vivo</i>. Chromatin Immunoprecipitation (ChIP) was performed using embryonic hearts stage ED10.5–11.0. PCR of the DNA input, Mef2c immunoprecipitate, and IgG control precipitate was performed using primers flanking both Mef2 consensus sites of the Crtl1 promoter. For the Sox9 ChIP, the initial DNA input, Sox9 immunoprecipitate, and IgG control precipitate were PCR amplified using primers specific for the Sox9 consensus site on the Crtl1 promoter.</p

Crtl1 promoter activity is regulated by Mef2c.

<p>Fold change in luciferase activity driven by approximately 1 kb of the Crtl1 promoter in the pGL3 luciferase reporter vector was assayed in fetal chicken VICs (A and B) and in NIH3T3 cells (C and D). In fetal chicken VICs (A) and NIH3T3 cells (C), Crtl1 promoter activity was significantly increased with increasing concentrations of Mef2c. (B) Crtl1 promoter activity in the presence of 100 ng Mef2c with the addition of 200 ng of the Mef2-Engrailed dominant negative expression construct resulted in an approximately 30% reduction in Crtl1 reporter activity. (D) Mutations were introduced into the Crtl1 promoter construct at Mef2 Site 1 and Mef2 Site 2 (Crtl1-Mutant 1 and Crtl1-Mutant 2 respectively). Crtl1-Mutant 1 results in an approximately 30% reduction in Crtl1 promoter activation in the presence of 100 ng of Mef2c and Crtl1-Mutant 2 results in an approximately 50% reduction of Crtl1 activity.(*p<0.05, #p<0.1).</p