Probes for INS

PURPOSE: Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. ROP occurs as a consequence of postnatal hyperoxia exposure in premature infants, resulting in vasoproliferation in the retina. The tetraspan protein epithelial membrane protein-2 (EMP2) is highly expressed in the retinal pigment epithelium (RPE) in adults, and it controls vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line. We, therefore, hypothesized that Emp2 knockout (Emp2 KO) protects against neovascularization in murine oxygen-induced retinopathy (OIR). METHODS: Eyes were obtained from wildtype (WT) and Emp2 KO mouse pups at P7, P12, P17, and P21 after normoxia or hyperoxia (P7-P12) exposure. Following hyperoxia exposure, RNA sequencing was performed using the retina/choroid layers obtained from WT and Emp2 KO at P17. Retinal sections from P7, P12, P17, and P21 were evaluated for Emp2, hypoxia-inducible factor 1? (Hif1?), and VEGF expression. Whole mount images were generated to assess vaso-obliteration at P12 and neovascularization at P17. RESULTS: Emp2 KO OIR mice demonstrated a decrease in pathologic neovascularization at P17 compared with WT OIR mice through evaluation of retinal vascular whole mount images. This protection was accompanied by a decrease in Hif1? at P12 and VEGFA expression at P17 in Emp2 KO animals compared with the WT animals in OIR conditions. Collectively, our results suggest that EMP2 enhances the effects of neovascularization through modulation of angiogenic signaling. CONCLUSIONS: The protection of Emp2 KO mice against pathologic neovascularization through attenuation of HIF and VEGF upregulation in OIR suggests that hypoxia-induced upregulation of EMP2 expression in the neuroretina modulates HIF-mediated neuroretinal VEGF expression

Defining the relationship between vascular development and the expression of hypoxia-inducible factors (Hifs) and vascular endothelial growth factor (Vegf) in the auditory brainstem is important to understand how tissue hypoxia caused by oxygen shortage contributes to sensory deficits in neonates. In this study, we used histology, molecular labeling, confocal microscopy and 3D image processing methods to test the hypothesis that significant maturation of the vascular bed in the medial nucleus of the trapezoid body (MNTB) occurs during the postnatal period that precedes hearing onset. Isolectin-B4 histochemistry experiments suggested that the MNTB vasculature becomes more elaborate between P5 and P10. When combined with a cell proliferation marker and immunohistochemistry, we found that vascular growth coincides with a switch in the localization of proliferating cells to perivascular locations, and an increase in the density of microglia within the MNTB. Furthermore, microglia were identified as perivascular cells with proliferative activity during the period of vascular maturation. Lastly, combined in situ hybridization and immunohistochemistry experiments showed distinct profiles of Hif1a and Vegf mRNA localization in microglia, astrocytes and MNTB principal neurons. These results suggest that different cells of the neuro-glio-vascular unit are likely targets of hypoxic insult in the auditory brainstem of neonate rats.

Research on human nail tissue has been limited by the restricted access to fresh specimen. Here, we studied transcriptome profiles of human nail units using polydactyly specimens. Single-cell RNAseq with 11,541 cells from 4 extra digits revealed nail-specific mesenchymal and epithelial cell populations, characterized by RSPO4 (major gene in congenital anonychia) and SPINK6, respectively. In situ RNA hybridization demonstrated the localization of RSPO4, MSX1 and WIF1 in onychofibroblasts suggesting the activation of WNT signaling. BMP-5 was also expressed in onychofibroblasts implicating the contribution of BMP signaling. SPINK6 expression distinguished the nail-specific keratinocytes from epidermal keratinocytes. RSPO4+ onychofibroblasts were distributed at close proximity with LGR6+ nail matrix, leading to WNT/β-catenin activation. In addition, we demonstrated RSPO4 was overexpressed in the fibroblasts of onychomatricoma and LGR6 was highly expressed at the basal layer of the overlying epithelial component, suggesting that onychofibroblasts may play an important role in the pathogenesis of onychomatricoma.

During gestation, the most drastic change in oxygen supply occurs with the onset of ventilation after birth. As the too early exposure of premature infants to high arterial oxygen pressure leads to characteristic diseases, we studied the adaptation of the oxygen sensing system and its targets, the hypoxia-inducible factor- (HIF-) regulated genes (HRGs) in the developing lung. We draw a detailed picture of the oxygen sensing system by integrating information from qPCR, immunoblotting, in situ hybridization, and single-cell RNA sequencing data in ex vivo and in vivo models. HIF1α protein was completely destabilized with the onset of pulmonary ventilation, but did not coincide with expression changes in bona fide HRGs. We observed a modified composition of the HIF-PHD system from intrauterine to neonatal phases: Phd3 was significantly decreased, while Hif2a showed a strong increase and the Hif3a isoform Ipas exclusively peaked at P0. Colocalization studies point to the Hif1a-Phd1 axis as the main regulator of the HIF-PHD system in mouse lung development, complemented by the Hif3a-Phd3 axis during gestation. Hif3a isoform expression showed a stepwise adaptation during the periods of saccular and alveolar differentiation. With a strong hypoxic stimulus, lung ex vivo organ cultures displayed a functioning HIF system at every developmental stage. Approaches with systemic hypoxia or roxadustat treatment revealed only a limited in vivo response of HRGs. Understanding the interplay of the oxygen sensing system components during the transition from saccular to alveolar phases of lung development might help to counteract prematurity-associated diseases like bronchopulmonary dysplasia.

Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The in vivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.

Here, we ask why the nail base is essential for mammalian digit tip regeneration, focusing on the inductive nail mesenchyme. We identify a transcriptional signature for these cells that includes Lmx1b and show that the Lmx1b-expressing nail mesenchyme is essential for blastema formation. We use a combination of Lmx1bCreERT2-based lineage-tracing and single-cell transcriptional analyses to show that the nail mesenchyme contributes cells for two pro-regenerative mechanisms. One group of cells maintains their identity and regenerates the new nail mesenchyme. A second group contributes specifically to the dorsal blastema, loses their nail mesenchyme phenotype, acquires a blastema transcriptional state that is highly similar to blastema cells of other origins, and ultimately contributes to regeneration of the dorsal but not ventral dermis and bone. Thus, the regenerative necessity for an intact nail base is explained, at least in part, by a requirement for the inductive nail mesenchyme.