A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana.

Root architecture is a major determinant of plant fitness and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can alter the position, spacing, density, and length of secondary or lateral roots. Lateral root development is among the best-studied examples of plant organogenesis, yet there are still many unanswered questions about its earliest steps. Among the challenges faced in capturing these first molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. Single-cell sequencing methods afford the opportunity to isolate the specific transcriptional changes occurring in cells undergoing this fate transition. Using this approach, we successfully captured the transcriptomes of initiating lateral root primordia in Arabidopsis thaliana and discovered many upregulated genes associated with this process. We developed a method to selectively repress target gene transcription in the xylem pole pericycle cells where lateral roots originate and demonstrated that the expression of several of these targets is required for normal root development. We also discovered subpopulations of cells in the pericycle and endodermal cell files that respond to lateral root initiation, highlighting the coordination across cell files required for this fate transition.

Anti-VEGF antibody leads to later atypical intravitreous neovascularization and activation of angiogenic pathways in a rat model of retinopathy of prematurity.

PURPOSE: Inhibiting VEGF improves adult retino/choroido-vascular diseases, but can lead to recurrent intravitreous neovascularization (IVNV), avascular retina (AVA), and retinal detachment in preterm infants with retinopathy of prematurity (ROP). We sought to understand causes of late-onset IVNV and AVA following anti-VEGF using an ROP model. METHODS: In the Penn model of ROP, postnatal day (p)12 pups received 1 µL intravitreal VEGFA164 antibody (anti-VEGF; 25-100 ng) or IgG control in each eye. Analyses included lectin-stained percent IVNV and AVA; VEGF protein, erythropoietin, phosphorylated extracellular signal-related kinases and signal transducer and activator of transcription-3 (p-STAT3); and immunohistochemistry of retinal sections for p-VEGFR2. Western blots of human retinal microvascular endothelial cells (hRMVECs) stimulated with VEGF or erythropoietin were analyzed for p-STAT3. Statistical analysis was performed with one-way ANOVA or two-tailed t-tests. RESULTS: At p18, 50 ng anti-VEGF reduced IVNV, and at p25, caused increased IVNV and AVA compared with controls. VEGF and p-VEGFR2 labeling increased following 100 ng anti-VEGF. Following 50 ng anti-VEGF, reduced p-STAT3 and increased erythropoietin occurred at p18. Erythropoietin or VEGF stimulated hRMVEC proliferation and STAT3 activation. In vivo, anti-VEGF reduced pup growth. CONCLUSIONS: Increases in erythropoietin and angiogenic signaling following anti-VEGF may account for recurrent IVNV. Anti-VEGF reduced pup growth. Research is needed regarding safety, dose, and type of antiangiogenic treatment for ROP.

VEGF-mediated STAT3 activation inhibits retinal vascularization by down-regulating local erythropoietin expression.

Avascular, hypoxic retina has been postulated to be a source of angiogenic factors that cause aberrant angiogenesis and intravitreal neovascularization (IVNV) in retinopathy of prematurity. Vascular endothelial growth factor (VEGF) is an important factor involved. However, VEGF is also required for normal retinal vascular development, which raises concerns about inhibiting its activity to treat IVNV in retinopathy of prematurity. Therefore, understanding the effects that VEGF has on other factors in the development of avascular retina is important to prevent aberrant angiogenesis and IVNV. Here, we show that STAT3 was activated by increased retinal VEGF in the rat 50/10 oxygen-induced retinopathy model. Phospho-STAT3 colocalized with glutamine synthetase-labeled Müller cells. Inhibition of STAT3 reduced avascular retina and increased retinal erythropoietin (Epo) expression. Epo administered exogenously also reduced avascular retina in the model. In an in vitro study, hypoxia-induced VEGF inhibited Epo gene expression by STAT3 activation in rat Müller cells. The mechanism by which activated STAT3 regulated Epo was by inhibition of Epo promoter activity. Together, these findings show that increased retinal VEGF contributes to avascular retina by regulating retinal Epo expression through Janus kinase/STAT signaling. Our results suggest that rescuing Epo expression in the retina before the development of IVNV may promote normal developmental angiogenesis and, therefore, reduce the stimulus for later pathologic IVNV.