To advance our understanding of development, function and diseases in the kidney glomerulus, we have established and large-scale sequenced cDNA libraries from mouse glomeruli at different stages of development, resulting in a catalogue of 6053 different genes. The glomerular cDNA clones were arrayed and hybridized against a series of labeled targets from isolated glomeruli, non-glomerular kidney tissue, FACS-sorted podocytes and brain capillaries, which identified over 300 glomerular cell-enriched transcripts, some of which were further sublocalized to podocytes, mesangial cells and juxtaglomerular cells by in situ hybridization. For the earliest podocyte marker identified, Foxc2, knockout mice were used to analyze the role of this protein during glomerular development. We show that Foxc2 controls the expression of a distinct set of podocyte genes involved in podocyte differentiation and glomerular basement membrane maturation. The primary podocyte defects also cause abnormal differentiation and organization of the glomerular vascular cells. We surmise that studies on the other novel glomerulus-enriched transcripts identified in this study will provide new insight into glomerular development and pathomechanisms of disease.
Pericytes are associated with the abluminal wall of microvessels, ie, arterioles, venules, and capillaries. They can be distinguished from other perivascular cell types by their location within the microvascular basement membrane. Pericytes, such as vascular smooth muscle cells (VSMCs; the mural cells of larger vessel), are mesenchymal cells of presumed mesodermal or neuroectodermal origin.
RGS proteins finely tune heterotrimeric G-protein signaling. Implying the need for such fine-tuning in the developing vascular system, in situ hybridization revealed a striking and extensive expression pattern of Rgs5 in the arterial walls of E12.5-E17.5 mouse embryos. The distribution and location of the Rgs5-positive cells typified that of pericytes and strikingly overlapped the known expression pattern of platelet-derived growth factor receptor (PDGFR)-beta. Both E14.5 PDGFR-beta- and platelet-derived growth factor (PDGF)-B-deficient mice exhibited markedly reduced levels of Rgs5 in their vascular plexa and small arteries. This likely reflects the loss of pericytes in the mutant mice. RGS5 acts as a potent GTPase activating protein for Gi(alpha) and Gq(alpha) and it attenuated angiotensin II-, endothelin-1-, sphingosine-1-phosphate-, and PDGF-induced ERK-2 phosphorylation. Together these results indicate that RGS5 exerts control over PDGFR-beta and GPCR-mediated signaling pathways active during fetal vascular maturation.
Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of microvascular abnormalities associated with diabetes and tumors. However, the unambiguous identification of pericytes is still a problem because of cellular heterogeneity and few available molecular markers. Here we describe an approach to identify pericyte markers based on transcription profiling of pericyte-deficient brain microvessels isolated from platelet-derived growth factor (PDGF-B)-/- and PDGF beta receptor (PDGFRbeta)-/- mouse mutants. The approach was validated by the identification of known pericyte markers among the most down-regulated genes in PDGF-B-/- and PDGFRbeta-/- microvessels. Of candidates for novel pericyte markers, we selected ATP-sensitive potassium-channel Kir6.1 (also known as Kcnj8) and sulfonylurea receptor 2, (SUR2, also known as Abcc9), both part of the same channel complex, as well as delta homologue 1 (DLK1) for in situ hybridization, which demonstrated their specific expression in brain pericytes of mouse embryos. We also show that Kir6.1 is highly expressed in pericytes in brain but undetectable in pericytes in skin and heart. The three new brain pericyte markers are signaling molecules implicated in ion transport and intercellular signaling, potentially opening new windows on pericyte function in brain microvessels.
Platelet-derived growth factor (PDGF)- A–deficient male mice were found to develop progressive reduction of testicular size, Leydig cells loss, and spermatogenic arrest. In normal mice, the PDGF-A and PDGF-Rα expression pattern showed positive cells in the seminiferous epithelium and in interstitial mesenchymal cells, respectively. The testicular defects seen in PDGF-A−/− mice, combined with the normal developmental expression of PDGF-A and PDGF-Rα, indicate that through an epithelial-mesenchymal signaling, the PDGF-A gene is essential for the development of the Leydig cell lineage. These findings suggest that PDGF-A may play a role in the cascade of genes involved in male gonad differentiation. The Leydig cell loss and the spermatogenic impairment in the mutant mice are reminiscent of cases of testicular failure in man.
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