Directed expression of transgenes to alveolar type I cells in the mouse.

Podoplanin (RTI40, aggrus, T1alpha, hT1alpha-2, E11, PA2.26, RANDAM-2, gp36, gp38, gp40, OTS8) is a type I cell marker in rat lung. We show that a bacterial artificial chromosome vector containing the rat podoplanin gene (RTIbac) delivers a pattern of transgene expression in lung that is more restricted to mouse type I cells than that of the endogenous mouse podoplanin gene. RTIbac-transgenic mice expressed rat podoplanin in type I cells; type II cells, airways, and vascular endothelium were negative. A modified bacterial artificial chromosome containing internal ribosome entry site (IRES)-green fluorescent protein (GFP) sequences in the podoplanin 3'UTR expressed rat podoplanin and transgenic GFP in type I cells. RTIbac transgene expression was absent or reduced in pulmonary pleura, lymphatic endothelium, and putative lymphoid-associated stromal tissue, all of which contained abundant mouse podoplanin. Rat podoplanin mRNA levels in normal rat lung and RTIbac transgenic lung were 25-fold higher than in corresponding kidney and brain samples. On Western blots, transgenic rat and endogenous mouse podoplanin displayed very similar patterns of protein expression in various organs. Highest protein levels were observed in lung with 10- to 20-fold less in brain; there were low levels in thymus and kidney. Both GFP and rat podoplanin transgenes were expressed at extrapulmonary sites of endogenous mouse podoplanin gene expression, including choroid plexus, eye ciliary epithelium, and renal glomerulus. Because their pulmonary expression is more restricted than endogenous mouse podoplanin, RTIbac derivatives should be useful for mouse type I cell-specific transgene delivery.

Genetic analysis of congenital cystic adenomatoid malformation reveals a novel pulmonary gene: fatty acid binding protein-7 (brain type).

The pathogenesis of congenital cystic adenomatoid malformation (CCAM) is unknown and its natural history is unpredictable. Fatty acid binding protein-7 (FABP-7) has been previously described in brain and breast development, but never before in the lung. We investigate gene expression in CCAM, and hypothesize that CCAM results from an aberration in the signaling pathway during lung development. Under IRB approval, tissue specimens of fetal CCAM, fetal control, postnatal CCAM, and postnatal control were examined and microarray analysis was performed. Candidate differentially expressed genes were selected with log-odds ratio (B) >0 and false discovery rate <0.05. Validation of differential expression was achieved at the RNA and protein levels. FABP-7 was underexpressed in fetal CCAM compared with fetal lung in both the microarray and by RT-PCR. Findings were duplicated by Western Blot analysis and immunohistochemistry. This is the first description of FABP-7 in the human lung. Decreased expression of FABP-7 in fetal CCAM compared with normal fetal lung at both the RNA and protein levels suggests FABP-7 may have a role in pulmonary development and in the pathogenesis of CCAM.

Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes.

We used microarray analysis with Affymetrix rat chips to determine gene expression profiles of freshly isolated rat type I (TI) and TII cells and cultured TII cells. Our goals were 1) to describe molecular phenotypic "fingerprints" of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about this cell type, and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. For these experiments, we used preparations of isolated TI and TII cells that contained <2% cross-contamination. With a false discovery rate of 1%, 601 genes demonstrated over twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about functions of TI and TII cells. We found similar numbers of differentially expressed genes between freshly isolated TI or TII cells and cultured TII cells (698, 637 genes) and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference including cluster dendrograms and log/log identity plots indicated major differences between the phenotypes of freshly isolated TI cell and cultured type II cell populations. The latter results suggest that experiments with TII cells cultured under these conditions should be interpreted with caution with respect to biological relevance to TI or TII cells.

Cerebral lobes in autism: early hyperplasia and abnormal age effects.

Metabolic, functional, behavioral, and histologic studies suggest that the structure of the cerebrum may be abnormal in autism. In a previous cross-sectional study we found abnormal enlargement of cerebral cortex and cerebral white matter volumes in autistic 2- and 3-year-olds and abnormally slow rates of volume change across later ages. In the present study, we assessed whether these volume abnormalities are limited to particular cerebral regions or are pervasive throughout the cerebrum. We used magnetic resonance imaging (MRI) to quantify volumes of cerebral lobes (frontal, temporal, parietal, and occipital regions), using classic sulcal boundaries to define regions. We examined 38 boys with autism and 39 normal control boys between the ages of 2 and 11 years. Several regions showed signs of gray matter and white matter hyperplasia in 2- and 3-year-old patients (as much as 20% enlargement), but there appeared to be an anterior to posterior gradient in the degree of hyperplasia. The frontal lobe showed the greatest enlargement while the occipital lobe was not significantly different from normal. Gray and white matter differences were not found in the older children. By examining the relationships between regional volumes and subject age, we found that frontal, temporal, and parietal white matter volumes, as well as frontal and temporal gray matter volumes, changed at significantly slower rates in autism patients than in controls across the 2- to 11-year-age range. For example, frontal lobe white matter volume increased by about 45% from 2-4 years of age to 9-11.5 years, but by only 13% in autistic patients. Mechanisms that might account for early hyperplasia are discussed as they might relate to the regional differences in degree of abnormality. For instance, possible influences of neurotrophic factors, or of abnormal afferent activity from other affected brain regions are considered.

Identification of genes differentially expressed in rat alveolar type I cells.

Although approximately 98% of the internal surface area of the lung is lined by alveolar type I cells, little is known about the functions of this cell type. Using freshly isolated rat type I and type II cells, we created a subtraction library by suppression subtractive hybridization to identify genes differentially expressed by type I cells. We identified twelve genes of known function that are differentially expressed by type I cells. Differential expression of all 12 genes was confirmed by Northern blotting; we confirmed differential expression by immunocytochemistry for 3 genes for which suitable antibodies were available. Most of the genes code for proteins that are multifunctional. From the known functions of these genes, we infer that type I cells may play a role in the maintenance of normal alveolar homeostasis and protection from injury, lung development and remodeling, host defense, tumor/growth suppression, and surfactant metabolism, among other functions.