Margaretha Jacobson

Architecture of apical dendrites in the murine neocortex: Dual apical dendritic systems

A monoclonal antibody (5F9) against microtubule-associated protein 2 is a selective and sensitive marker for neocortical dendrites in the mouse. The marker stains all dendrites. It affords a particularly comprehensive picture of the patterns of arrangements of apical dendrites which are most intensely stained with this antibody. Dual systems of apical dendrites arise from the polymorphic neurons of layer VI, on the one hand, and the pyramidal neurons of layers II-V, on the other. Terminal arborization of the former is concentrated principally at the interface of layers V and IV, while that of the latter is in the molecular layer. Apical dendrites of both systems are grouped into fascicles. In supragranular layers and in upper layer VI-lower layer V, where apical dendrites are most abundant, the fascicles coalesce into septa. These generate a honeycomb-like pattern, subdividing these cortical levels into columnar spaces of approximately 20-40 micron diameter. At the level of layer IV, where the number of apical dendrites is greatly reduced, the fascicles are isolated bundles. These bundles have the form of circular, elliptical or rectangular columns in the primary somatosensory, temporal and frontal regions, respectively. Those in the barrel field are preferentially concentrated in the sides of barrels and the interbarrel septa. The configurations of the dendritic fascicles, particularly the midcortical bundles, may conform to the spatial configuration of investing axons of interneurons.

δ-catenin is a nervous system-specific adherens junction protein which undergoes dynamic relocalization during development

δ‐catenin is a member of the Armadillo repeat family and component of the adherens junction discovered in a two‐hybrid assay as a bona fide interactor with presenilin‐1 (Zhou et al., [ 1997 ], NeuroReport 8:2085–2090), a protein which carries mutations that cause familial Alzheimers disease. The expression pattern of δ‐catenin was mapped between embryonic day 10 (E10) and adulthood by Northern blots, in situ hybridization and immunohistochemistry in the mouse. In development, δ‐catenin is dynamically regulated with respect to its site of expression. It is first expressed within proliferating neuronal progenitor cells of the neuroepithelium, becomes down‐regulated during neuronal migration, and is later reexpressed in the dendritic compartment of postmitotic neurons. In the mouse, δ‐catenin mRNA is expressed by E10, increases and peaks at postnatal day (P)7, with lower levels in adulthood. In the developing neocortex, δ‐catenin mRNA is strongly expressed in the proliferative ventricular zone and the developing cortical plate, yet is conspicuously less prominent in the intermediate zone, which contains migrating cortical neurons, δ‐catenin protein forms a honeycomb pattern in the neuroepithelium by labeling the cell periphery in a typical adherens junction pattern. By E18, δ‐catenin expression shifts primarily to nascent apical dendrites, a pattern that continues through adulthood. The dynamic relocalization of δ‐catenin expression during development, taken together with previously published data which described a role for δ‐catenin in cell motility (Lu et al., [ 1999 ] J. Cell. Biol. 144:519–532), suggests the hypothesis that δ‐catenin regulation is closely linked to neuronal migration and may play a role in the establishment of mature dendritic relationships in the neuropil. J. Comp. Neurol. 420:261–276, 2000.

Ontogenesis of microtubule-associated protein 2 (MAP2) in embryonic mouse cortex.

The developing neocortex in mice from embryonic day 13 (E13) until birth (E19) was immunoreacted with a monoclonal antibody for microtubule-associated protein 2 (MAP2) that is highly specific for neuronal somata and dendrites. In E13 neocortex there was no detectable MAP2 immunoreactivity on tissue sections or on gel blots. From E14 to birth the MAP2 immunoreactivity was present in both tissue sections and immunoblots of homogenized cortex. In the neocortex the staining pattern was lamina-specific. The molecular layer and the cortical subplate contained the most dense staining of dendrites and cell somata. The cortical plate showed weak to moderate staining at these ages while the intermediate and ventricular zones were not stained above background control levels. Gel blots correspondingly did not show detectable levels of MAP2 until E14. Ultrastructural data suggest that MAP2 is present in dendrites in each of the laminae. The laminar pattern of MAP2 immunoreactivity may be due to either the higher density of differentiating dendrites in the molecular and subplate layers or to compartmentalization of MAP2 within individual cortical neurons.

Expression of nitric oxide synthase isoforms (NOS II and NOS III) in adult rat lung in hyperoxic pulmonary hypertension.

Abstract Breathing air with a high oxygen tension induces an inflammatory response and injures the microvessels of the lung. The resulting development of smooth muscle cells in these segments contributes to changes in vasoreactivity and increased pulmonary artery pressure. This in vivo study determines the temporal and spatial expression of endogenous endothelial nitric oxide synthase (NOS III) and inducible NOS (NOS II), important enzymes regulating vasoreactivity and inflammation, in the adult rat lung during the development of experimental pulmonary hypertension induced by oxidant injury. We analyzed the cellular distribution of these NOS isoforms, using specific antibodies, and assessed enzyme activity at baseline and after 1–28 days of hyperoxia (FIO2 0.87). The number of NOS III-immuno-positive endothelial cells increased early in hyperoxia and then remained high. By day 28, the relative number of these cells had increased from 40% in proximal vessels and 13–16% in distal alveolar vessels of the normal lung to 73–86% and 40–59%, respectively, in hyperoxia. Pulmonary alveolar macrophages (PAMs), normally few in number and only weakly immunopositive for NOS II or III in the normal lung, increased in number in hyperoxia and were strongly immunopositive for each isoform. These morphological data were supported by a temporal increase in total and calcium-independent NOS activity. Thus NOS expression and activity significantly increased in hyperoxia as pulmonary hypertension developed, and NOS III expression increased selectively in vascular endothelial cells, while both NOS isoforms were expressed by the PAM population. We conclude that this increase in expression of a potent vasodilator, an antiproliferative agent for smooth muscle cells, and an antioxidant molecule represents an adaptive response to protect the lung from oxidant-induced vascular and epithelial injury.

Microvessel precursor smooth muscle cells express head-inserted smooth muscle myosin heavy chain (SM-B) isoform in hyperoxic pulmonary hypertension.

Abstract The present study analyzes smooth muscle myosin heavy chain (SMMHC) expression as lung microvascular precursor smooth muscle cells (PSMCs), cells derived from fibroblasts and intermediate cells (immature SMCs), acquire a smooth muscle phenotype in anin vivo model of pulmonary hypertension (PH). Because of the unique contractile properties of the SMMHC isoform SM-B, we analyzed its expression in the microvessels (<100 μm diameter) and in larger vessels (100–700 μm) quantitatualy by the labeled [strept]avidin-biotin technique (day 1–28), and related this to cell phenotype by transmission microscopy and protein A-gold labeling (at day 28). Airway SMCs of the normal and hypertensive lung uniformly expressed SM-B whereas vascular SMC expression was heterogeneous. Thus, in some large arteries (and veins) SMCs contained cells expressing SM-B while in others all the cells were immunonegative. Microvascular cells expressing SM-B (arteries and veins) were rare in normal lung and numerous in PH, increasing as wall muscle developed in smaller segments with time. As in large vessels, some microvessels had immunopositive cells and others only negative ones. Ultrastructural analysis confirmed that the SMCs of bronchial vessels, and the septal SMCs adjoining alveolar ducts, contained dense filament arrays decorated with SM-B. While the PSMC processes of the normal lung contained sparse filaments decorated with SM-B, these cells expressed dense filament arrays in PH. Fibroblasts migrating to align around the microvessels also expressed SM-B but in the absence of a filament network. For the first time,we demonstrate in vivo that newly developed microvascular PSMCs express the SMMHC SM-B isoform in PH.

PDGF and Microvessel Wall Remodeling in Adult Lung: Imaging PDGF-Rβ and PDGF-BB Molecules in Progenitor Smooth Muscle Cells Developing in Pulmonary Hypertension

Smooth muscle cells are relatively rare cells in the microvessels of the normal adult lung but develop in high numbers in the clinical pulmonary hypertensions (PHs). Understanding this cellular response has profound implications for determining the pathogenesis of PH, and for the development of therapeutic strategies, yet little is known of the angiogenic molecules responsible. The authors have previously shown that interstitial fibroblasts, and intermediate cells, are the progenitors of smooth muscle cells developing in adult lung microvessels in an in vivo model of experimental PH. The present study evaluates PDGF-Rβ/PDGF-BB, an important angiogenic signaling pathway, using antibodies linked to protein A-gold (pA-AU) and quantitative high-resolution imaging techniques to detect expression by these cells. Each progenitor cell type in the control lung expressed PDGF-Rβ and PDGF-BB. In the hypertensive lung, PDGF-Rβ was highly expressed by fibroblasts developing as perivascular cells, the mean number of pA-AU labeled antigenic sites per cell profile, and their density (μm−2), increasing with time: in intermediate cells the mean number of sites per cell profile, although not their density (μm−2), also increased with time but less so than in the fibroblasts. In clear contrast to the RTK, constitutive expression levels of PDGF-BB were low in each progenitor cell type and remained restricted in the hypertensive lung.

PDGF and microvessel wall remodeling in adult rat lung: imaging PDGF-AA and PDGF-Rα molecules in progenitor smooth muscle cells developing in experimental pulmonary hypertension

Smooth muscle cells are mostly absent from the walls of microvessels in the adult lung but develop in large numbers as part of the pathology of human and experimental pulmonary hypertensions (PHs). We have previously shown, in an in vivo model of experimental PH, that mesenchymal (interstitial) fibroblasts and intermediate cells are the progenitors of these cells. Although smooth muscle cell development is a defining pathophysiological feature of human PH, little is known about the angiogenic signaling molecules responsible. Here, we report data for platelet-derived growth factor AA (PDGF-AA) and PDGF-Rα, two components of an important signaling pathway for fibroblast and myofibroblast proliferation and migration. Using antibodies linked to protein-A gold and high-resolution imaging techniques, we analyzed the expression of these molecules as smooth muscle cells developed from progenitor cell populations and in endothelial cells of the same microvessels. PDGF-AA was highly expressed by each cell type in control lung. As PH developed, the number of antigenic sites for PDGF-AA decreased with time. PDGF-Rα expression levels in the control lung were low, relative to the ligand, and fell in PH. These data show, for the first time, a marked phenotypic shift in expression levels of the PDGF-AA isoform and its receptor tyrosine kinase in the progenitor smooth muscle cells developing in the microvessels of the adult hypertensive lung.

VEGFR2+PDGFRβ+ circulating precursor cells participate in capillary restoration after hyperoxia acute lung injury (HALI)

The in vivo morphology and phenotype of circulating cells that spontaneously contribute to new vessel formation in adults remain unclear. Here, we use high‐resolution imaging and flow cytometry to characterize the morphology and phenotype of a distinct population of circulating mononuclear cells contributing to spontaneous new vessel formation after hyperoxia acute lung injury (HALI). We identify a subpopulation of myeloid (CD11b/Mac1+) haematopoietic cells co‐expressing vascular endothelial growth factor receptor 2 (VEGFR2) and platelet derived growth factor receptor beta (PDGFRβ). Moreover, we show that these CD11b+VEGFR2+PDGFRβ+ circulating precursor cells (CPCs) contribute structurally to the luminal surface of capillaries re‐forming 2 weeks post‐HALI. This indicates that these myeloid CPCs may function, at least transiently, as putative vascular precursors, and has important implications for capillary growth and repair in injury and in pathologies of the lung and other organs.