John M. Shannon

Development of definitive endoderm from embryonic stem cells in culture

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury+ population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury+ cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.

Nonselenium Glutathione Peroxidase in Human Brain: Elevated Levels in Parkinson's Disease and Dementia with Lewy Bodies

Nonselenium glutathione peroxidase (NSGP) is a new member of the antioxidant family. Using antibodies to recombinant NSGP we have examined the distribution of this enzyme in normal, Parkinsons disease (PD), and dementia with Lewy body disease (DLB) brains. We have also co-localized this enzyme with alpha-synuclein as a marker for Lewy bodies. In normal brains there was a very low level of NSGP staining in astrocytes. In PD and DLB there were increases in the number and staining intensity of NSGP-positive astrocytes in both gray and white matter. Cell counting of NSGP cells in PD and DLB frontal and cingulated cortices indicated there was 10 to 15 times more positive cells in gray matter and three times more positive cells in white matter than in control cortices. Some neurons were positive for both alpha-synuclein and NSGP in PD and DLB, and double staining indicated that NSGP neurons contained either diffuse cytoplasmic alpha-synuclein deposits or Lewy bodies. In concentric Lewy bodies, alpha-synuclein staining was peripheral whereas NSGP staining was confined to the central core. Immunoprecipitation indicated there was direct interaction between alpha-synuclein and NSGP. These results suggest oxidative stress conditions exist in PD and DLB and that certain cells have responded by up-regulating this novel antioxidant enzyme.

BMP4 modulates fibroblast growth factor–mediated induction of proximal and distal lung differentiation in mouse embryonic tracheal epithelium in mesenchyme‐free culture

Lung morphogenesis and differentiation require interaction between the epithelium and mesenchyme, which is mediated by diffusible molecules such as fibroblast growth factors (FGFs), bone morphogenetic protein 4 (BMP4), and Shh. We have used mesenchyme‐free culture to study the effects of these molecules on lung epithelial differentiation. We have tested the individual abilities of FGF1, FGF2, FGF7, FGF9, FGF10, and FGF18, as well as BMP4 and Shh to promote growth and specify distal lung differentiation in mouse tracheal epithelium. The different FGFs exhibited distinct abilities to induce epithelial growth and the expression of the distal lung epithelial marker, surfactant protein C (SP‐C), although all FGFs were able to induce expression of BMP4. Tracheal epithelium treated with FGF10 showed little growth and failed to express SP‐C as measured by whole‐mount in situ hybridization and quantitative real‐time polymerase chain reaction. FGF1 treatment resulted in the strongest induction of SP‐C. Treatment with BMP4 inhibited epithelial growth and differentiation and antagonized the stimulatory effects of FGF1. In contrast, inhibition of endogenous BMP4 signaling with Noggin protein did not inhibit growth or expression of SP‐C but did increase the expression of the proximal lung markers CCSP and HFH4. Expression of Shh was not affected by any of the conditions tested. These results suggest that BMP4 does not signal epithelial cells to adopt a distal fate but may regulate the expansion of proximal epithelial cells in the lung.

Expression of a brain-type cannabinoid receptor (CB1) in alveolar Type II cells in the lung: regulation by hydrocortisone.

Using the polymerase chain reaction with degenerate primers to identify novel G-protein-coupled receptors of the rat alveolar Type II cell, we identified sequences expressed by the Type II cell identical to the sequence of the rat brain cannabinoid receptor (CB1). The use of Northern blot analysis to examine expression of CB1 mRNA in rat tissues revealed differences between the brain and lung. While rat brain expressed a 6.0 kb mRNA as previously described, rat lung expressed mRNA of 4.5 and 6.0 kb. Isolated lung alveolar Type II cells also expressed mRNA of 4.5 and 6.0 kb as determined by Northern analysis. However, only freshly isolated Type II cells contained cannabinoid receptor mRNA. Reverse transcriptase-polymerase chain reaction (RT-PCR) failed to detect CB1 mRNA in Type II cells maintained in culture for 1 or 2 days. We next determined developmental changes in lung CB1 mRNA expression using semi-quantitative RT-PCR. CB1 expression was detected as early as gestational day 16 in rat lung and mRNA levels increased to fetal day 20 before birth, before declining to adult levels. Fetal rat lung explants were utilized to further examine the ontogeny and hormonal effects on CB1 mRNA expression. Hydrocortisone induced a dose-dependent expression in 15-day and 18-day explants, similar to previous results for surfactant-associated proteins. Our results demonstrate expression of CB1 mRNA in rat alveolar Type II cells and rat lung. This expression is ontogenically and hormonally regulated, with maximal expression noted just prior to birth in rat lung. Since CB1 mRNA is only expressed in freshly isolated Type II cells, CB1 may be useful as a Type II cell marker.