GFP Mouse Research Articles

Uniform Distribution of Epithelial Stem Cells in the Bulbar Conjunctiva

To determine the distribution of epithelial stem cells in the bulbar conjunctiva by measuring homeostatic movements and mitosis of epithelial cells in this region. The ubiquitous GFP mouse was used to monitor movement of conjunctival epithelial cells. Cell movement was determined by histology, analyzing the shape and distribution of GFP cell clusters in flat wholemount specimens, and by in vivo time-lapse microscopy, tracking the movement of GFP-positive cells in the bulbar conjunctiva near the limbus. Mitoses were determined by labeling DNA of adult mice with bromodeoxyuridine (BrdU) for 3 days. Label-retaining cells (LRCs) were determined by a pulse label of newborn mice with BrdU, followed by a chase of 6 to 7 weeks. Similar to the corneal epithelium, only some of the conjunctival epithelial cells expressed a high level of GFP. Histology showed that GFP-positive cells existed as clusters of several to several dozen cells. No stripe pattern of GFP was observed in any part of the conjunctiva, suggesting that directed cell movement was rare or nonexistent. Time-lapse analyses revealed that none of the tracked GFP clusters exhibited a continuous and directed movement and that most GFP clusters were stationary for several weeks and much longer in some occasions. BrdU labeling showed that GFP-positive cells in this region were mitotically active. BrdU pulse-chase experiments demonstrated that LRCs were distributed uniformly in this region. Epithelial cells of the bulbar conjunctiva near the limbus are mitotically active and yet they are generally immobile in a lateral direction, indicating that these cells are self-sufficient. These results, combined with the uniform distribution of LRCs, suggest that epithelial stem cells are distributed uniformly in this area.

Injury of the renal microvascular endothelium alters barrier function after ischemia.

The role of renal microvascular endothelial cell injury in the pathophysiology of ischemic acute renal failure (ARF) remains largely unknown. No consistent morphological alterations have been ascribed to the endothelium of the renal microvasculature as a result of ischemia-reperfusion injury. Therefore, the purpose of this study was to examine biochemical markers of endothelial injury and morphological changes in the renal microvascular endothelium in a rodent model of ischemic ARF. Circulating von Willebrand factor (vWF) was measured as a marker of endothelial injury. Twenty-four hours after ischemia, circulating vWF peaked at 124% over baseline values (P = 0.001). The FVB-TIE2/GFP mouse was utilized to localize morphological changes in the renal microvascular endothelium. Immediately after ischemia, there was a marked increase in F-actin aggregates in the basal and basolateral aspect of renal microvascular endothelial cells in the corticomedullary junction. After 24 h of reperfusion, the pattern of F-actin staining was more similar to that observed under physiological conditions. In addition, alterations in the integrity of the adherens junctions of the renal microvasculature, as demonstrated by loss of localization in vascular endothelial cadherin immunostaining, were observed after 24 h of reperfusion. This observation temporally correlated with the greatest extent of permeability defect in the renal microvasculature as identified using fluorescent dextrans and two-photon intravital imaging. Taken together, these findings indicate that renal vascular endothelial injury occurs in ischemic ARF and may play an important role in the pathophysiology of ischemic ARF.

CUMMINS TO KEEP KUSS FILTRATION

Fluorescent proteins are commonly used to label cells across organisms, but the unmodified forms cannot control biological activities. Using GFP-binding proteins derived from Camelid antibodies, we co-opted GFP as a scaffold for inducing formation of biologically active complexes, developing a library of hybrid transcription factors that control gene expression only in the presence of GFP or its derivatives. The modular design allows for variation in key properties such as DNA specificity, transcriptional potency, and drug dependency. Production of GFP controlled cell-specific gene expression and facilitated functional perturbations in the mouse retina and brain. Further, retrofitting existing transgenic GFP mouse and zebrafish lines for GFP-dependent transcription enabled applications such as optogenetic probing of neural circuits. This work establishes GFP as a multifunctional scaffold and opens the door to selective manipulation of diverse GFP-labeled cells across transgenic lines. This approach may also be extended to exploit other intracellular products as cell-specific scaffolds in multicellular organisms.