Ebf1 is a mouse deafness gene and deletion causes a dramatic increase in hair cells and support cells of the organ of Corti.

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage prior to development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had no detectable auditory brainstem response (ABR) suggesting that this gene is essential for hearing development.

Novel cell types and developmental lineages revealed by single-cell RNA-seq analysis of the mouse crista ampullaris.

This study provides transcriptomic characterization of the cells of the crista ampullaris, sensory structures at the base of the semicircular canals that are critical for vestibular function. We performed single-cell RNA-seq on ampullae microdissected from E16, E18, P3, and P7 mice. Cluster analysis identified the hair cells, support cells and glia of the crista as well as dark cells and other nonsensory epithelial cells of the ampulla, mesenchymal cells, vascular cells, macrophages, and melanocytes. Cluster-specific expression of genes predicted their spatially restricted domains of gene expression in the crista and ampulla. Analysis of cellular proportions across developmental time showed dynamics in cellular composition. The new cell types revealed by single-cell RNA-seq could be important for understanding crista function and the markers identified in this study will enable the examination of their dynamics during development and disease.

STAT Signaling Modifies Ascl1 Chromatin Binding and Limits Neural Regeneration from Muller Glia in Adult Mouse Retina.

Müller glia (MG) serve as sources for retinal regeneration in non-mammalian vertebrates. We find that this process can be induced in mouse MG, after injury, by transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are generated only from a subset of MG. Identifying factors that limit Ascl1-mediated MG reprogramming could make this process more efficient. In this study, we test whether injury-induced STAT activation hampers the ability of Ascl1 to reprogram MG into retinal neurons. Single-cell RNA-seq shows that progenitor-like cells derived from Ascl1-expressing MG have a higher level of STAT signaling than do those cells that become neurons. Ascl1-ChIPseq and ATAC-seq show that STAT potentially directs Ascl1 to developmentally inappropriate targets. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of MG to generate neurons.

Open chromatin dynamics in prosensory cells of the embryonic mouse cochlea.

Hearing loss is often due to the absence or the degeneration of hair cells in the cochlea. Understanding the mechanisms regulating the generation of hair cells may therefore lead to better treatments for hearing disorders. To elucidate the transcriptional control mechanisms specifying the progenitor cells (i.e. prosensory cells) that generate the hair cells and support cells critical for hearing function, we compared chromatin accessibility using ATAC-seq in sorted prosensory cells (Sox2-EGFP+) and surrounding cells (Sox2-EGFP-) from E12, E14.5 and E16 cochlear ducts. In Sox2-EGFP+, we find greater accessibility in and near genes restricted in expression to the prosensory region of the cochlear duct including Sox2, Isl1, Eya1 and Pou4f3. Furthermore, we find significant enrichment for the consensus binding sites of Sox2, Six1 and Gata3-transcription factors required for prosensory development-in the open chromatin regions. Over 2,200 regions displayed differential accessibility with developmental time in Sox2-EGFP+ cells, with most changes in the E12-14.5 window. Open chromatin regions detected in Sox2-EGFP+ cells map to over 48,000 orthologous regions in the human genome that include regions in genes linked to deafness. Our results reveal a dynamic landscape of open chromatin in prosensory cells with potential implications for cochlear development and disease.

Effects of 3,3'-Iminodipropionitrile on Hair Cell Numbers in Cristae of CBA/CaJ and C57BL/6J Mice.

This study examines absolute hair cell numbers in the cristae of C57BL/6J mice and CBA/CaJ mice from weaning to adulthood as well as the dose required for 3,3'-iminodiproprionitrile (IDPN)-injury of the cristae in C57BL/6J mice and CBA/CaJ mice, the two mouse strains most commonly used by inner ear researchers. In cristae of CBA/CaJ and C57BL/6J mice, no loss of hair cells was observed up to 24 weeks. In both strains, dose-dependent loss of hair cells was observed 7 days after IDPN treatment of 2-month-old mice (IC50 = 16.1 mmol/kg in C57BL/6J mice vs. 25.21 mmol/kg in CBA/CaJ mice). Four-month-old C57BL/6J mice exposed to IDPN developed dose-dependent vestibular dysfunction as indicated by increased activity and circling behavior in open field tests and by failure to swim 7 days after treatment. IDPN-hair cell injury in C57BL/6J mice and CBA/CaJ mice represents a fast and predictable experimental model for the study of vestibular degeneration and a platform for the testing of vestibular therapies.

The role of sphingosine-1-phosphate in endothelial barrier function.

Loss of endothelial barrier function is implicated in the etiology of metastasis, atherosclerosis, sepsis and many other diseases. Studies suggest that sphingosine-1-phosphate (S1P), particularly HDL-bound S1P (HDL-S1P) is essential for endothelial barrier homeostasis and that HDL-S1P may be protective against the loss of endothelial barrier function in disease. This review summarizes evidence providing mechanistic insights into how S1P maintains endothelial barrier function, highlighting the recent findings that implicate the major S1P carrier, HDL, in the maintenance of the persistent S1P-signaling needed to maintain endothelial barrier function. We review the mechanisms proposed for HDL maintenance of persistent S1P-signaling, the evidence supporting these mechanisms and the remaining fundamental questions.

Cubilin maintains blood levels of HDL and albumin.

Cubilin is an endocytic receptor highly expressed in renal proximal tubules, where it mediates uptake of albumin and filtered forms of apoA-I/HDL. Cubilin deficiency leads to urinary loss of albumin and apoA-I; however, the consequences of cubilin loss on the homeostasis of blood albumin and apoA-I/HDL have not been studied. Using mice heterozygous for cubilin gene deletion (cubilin HT mice), we show that cubilin haploinsufficiency leads to reduced renal proximal tubular uptake of albumin and apoA-I and significantly increased urinary loss of albumin and apoA-I. Moreover, cubilin HT mice displayed significantly decreased blood levels of albumin, apoA-I, and HDL. The levels of albumin and apoA-I protein or mRNA expressed in the liver, kidney, or intestine of cubilin HT mice did not change significantly. The clearance rate of small HDL3 particles (density>1.13 g/ml) from the blood increased significantly in cubilin HT mice. In contrast, the rate of clearance of larger HDL2 particles from the blood did not change significantly, indicating a decreased half-life for HDL particles capable of filtering through the glomerulus. On the basis of these findings, we conclude that cubilin deficiency reduces renal salvage and delivery back to the blood of albumin and apoA-I, which decreases blood levels of albumin and apoA-I/HDL. These findings raise the possibility that therapeutic increase of renal cubilin expression might reduce proteinuria and increase blood levels of albumin and HDL.

Sphingosine 1-phosphate (S1P) carrier-dependent regulation of endothelial barrier: high density lipoprotein (HDL)-S1P prolongs endothelial barrier enhancement as compared with albumin-S1P via effects on levels, trafficking, and signaling of S1P1.

Sphingosine 1-phosphate (S1P) is a blood-borne lysosphingolipid that acts to promote endothelial cell (EC) barrier function. In plasma, S1P is associated with both high density lipoproteins (HDL) and albumin, but it is not known whether the carriers impart different effects on S1P signaling. Here we establish that HDL-S1P sustains EC barrier longer than albumin-S1P. We showed that the sustained barrier effects of HDL-S1P are dependent on signaling by the S1P receptor, S1P1, and involve persistent activation of Akt and endothelial NOS (eNOS), as well as activity of the downstream NO target, soluble guanylate cyclase (sGC). Total S1P1 protein levels were found to be higher in response to HDL-S1P treatment as compared with albumin-S1P, and this effect was not associated with increased S1P1 mRNA or dependent on de novo protein synthesis. Several pieces of evidence indicate that long term EC barrier enhancement activity of HDL-S1P is due to specific effects on S1P1 trafficking. First, the rate of S1P1 degradation, which is proteasome-mediated, was slower in HDL-S1P-treated cells as compared with cells treated with albumin-S1P. Second, the long term barrier-promoting effects of HDL-S1P were abrogated by treatment with the recycling blocker, monensin. Finally, cell surface levels of S1P1 and levels of S1P1 in caveolin-enriched microdomains were higher after treatment with HDL-S1P as compared with albumin-S1P. Together, the findings reveal S1P carrier-specific effects on S1P1 and point to HDL as the physiological mediator of sustained S1P1-PI3K-Akt-eNOS-sGC-dependent EC barrier function.

Reduction in postoperative high-density lipoprotein cholesterol levels in children undergoing the Fontan operation.

Despite the emerging relevance of high-density lipoprotein (HDL) in the inflammatory cascade and vascular barrier integrity, HDL levels in children undergoing cardiac surgery are unexplored. As a measure of HDL levels, the HDL-cholesterol (HDL-C) in single-ventricle patients was quantified before and after the Fontan operation, and it was determined whether relationships existed between the duration and the type of postoperative pleural effusions. The study prospectively enrolled 12 children undergoing the Fontan operation. Plasma HDL-C levels were measured before and after cardiopulmonary bypass. The outcome variables of interest were the duration and type of chest tube drainage (chylous vs. nonchylous). The Kendall rank correlation coefficient and the Wilcoxon rank sum test were used. There were 11 complete observations. The median preoperative HDL-C level for all the subjects was 30 mg/dl (range, 24-53 mg/dl), and the median postcardiopulmonary bypass level was 21 mg/dl (range, 14-46 mg/dl) (p = 0.004). There was a tendency toward a moderate inverse correlation (-0.42) between the postcardiopulmonary bypass HDL-C level and the duration of chest tube drainage, but the result was not statistically significant (p = 0.07). In the chylous effusion group, the median postcardiopulmonary bypass HDL-C tended to be lower (16 vs. 23 mg/dl; p = 0.09). After the Fontan operation, the plasma HDL-C levels in children are significantly reduced. It is reasonable to conclude that the reduction in HDL-C reflects reduced plasma levels of HDL particles, which may have pertinent implications in postoperative pleural effusions given the antiinflammatory and endothelial barrier functions of HDL.

S1P, dihydro-S1P and C24:1-ceramide levels in the HDL-containing fraction of serum inversely correlate with occurrence of ischemic heart disease.

BACKGROUND: The lysosphingolipid sphingosine 1-phosphate (S1P) is carried in the blood in association with lipoproteins, predominantly high density lipoproteins (HDL). Emerging evidence suggests that many of the effects of HDL on cardiovascular function may be attributable to its S1P cargo. METHODS: Here we have evaluated how levels of S1P and related sphingolipids in an HDL-containing fraction of human serum correlate with occurrence of ischemic heart disease (IHD). To accomplish this we used liquid chromatography-mass spectrometry to measure S1P levels in the HDL-containing fraction of serum (depleted of LDL and VLDL) from 204 subjects in the Copenhagen City Heart Study (CCHS). The study group consisted of individuals having high serum HDL cholesterol (HDL-C) (females:≥ 73.5 mg/dL; males:≥ 61.9 mg/dL) and verified IHD; subjects with high HDL-C and no IHD; individuals with low HDL-C (females:≤ 38.7 mg/dL; males:≤ 34.1 mg/dL) and IHD, and subjects with low HDL-C and no IHD. RESULTS: The results show a highly significant inverse relationship between the level of S1P in the HDL-containing fraction of serum and the occurrence of IHD. Furthermore, an inverse relationship with IHD was also observed for two other sphingolipids, dihydro-S1P and C24:1-ceramide, in the HDL-containing fraction of serum. Additionally, we demonstrated that the amount of S1P on HDL correlates with the magnitude of HDL-induced endothelial cell barrier signaling. CONCLUSIONS: These findings indicate that compositional differences of sphingolipids in the HDL-containing fraction of human serum are related to the occurrence of IHD, and may contribute to the putative protective role of HDL in IHD.

Sphingosine-1-phosphate signaling in vasculogenesis and angiogenesis.

Blood vessels either form de novo through the process of vasculogenesis or through angiogenesis that involves the sprouting and proliferation of endothelial cells in pre-existing blood vessels. A complex interactive network of signaling cascades downstream from at least three of the nine known G-protein-coupled sphingosine-1-phosphate (S1P) receptors act as a prime effector of neovascularization that occurs in embryonic development and in association with various pathologies. This review focuses on the current knowledge of the roles of S1P signaling in vasculogenesis and angiogenesis, with particular emphasis on vascular cell adhesion and motility responses.

High density lipoprotein-associated sphingosine 1-phosphate promotes endothelial barrier function.

High density lipoproteins (HDL) are major plasma carriers of sphingosine 1-phosphate (S1P). Here we show that HDL increases endothelial barrier integrity as measured by electric cell substrate impedance sensing. S1P was implicated as the mediator in this process through findings showing that pertussis toxin, an inhibitor of Gi-coupled S1P receptors, as well as antagonists of the S1P receptor, S1P1, inhibited barrier enhancement by HDL. Additional findings show that HDL stimulates endothelial cell activation of Erk1/2 and Akt, signaling pathway intermediates that have been implicated in S1P-dependent endothelial barrier activity. HDL was also found to promote endothelial cell motility, a process that may also relate to endothelial barrier function in the context of a vascular injury response. The effects of HDL on endothelial cell Erk1/2 and Akt activation and motility were suppressed by pertussis toxin and S1P1 antagonists. However, both HDL-induced barrier enhancement and HDL-induced motility showed a greater dependence on Akt activation as compared with Erk1/2 activation. Together, the findings indicate that HDL has endothelial barrier promoting activities, which are attributable to its S1P component and signaling through the S1P1/Akt pathway.

Sphingosine-1-phosphate signaling promotes critical migratory events in vasculogenesis.

Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of vasculogenesis in the mouse embryo. At stages preceding the formation of blood vessels (7.5-8 dpc) in the embryo proper, yolk sac, and allantois, the S1P receptor S1P(2) is expressed in conjunction with S1P(1) and/or S1P(3). Additionally, sphingosine kinase-2 (SK2), an enzyme that catalyzes the formation of S1P, is expressed in these tissues throughout periods of vasculogenesis. Using the cultured mouse allantois explant model of blood vessel formation, we found that vasculogenesis was dependent on S1P signaling. We showed that S1P could replace the ability of serum to promote vasculogenesis in cultured allantois explants. Instead of small poorly reticulated clusters of rounded endothelial cells that formed under serum-free conditions, S1P promoted the formation of elongated endothelial cells that arranged into expansive branched networks of capillary-like vessels. These effects could not be reproduced by vascular endothelial growth factor or basic fibroblast growth factor administration. The ability of S1P to promote blood vessel formation was not due to effects on cell survival or on changes in numbers of endothelial cells (Flk1(+)/PECAM(+)), angioblasts (Flk1(+)/PECAM(-)), or undifferentiated mesodermal cells (Flk1(-)/PECAM(-)). The S1P effect on blood vessel formation was attributed to it promoting migratory activities of angioblasts and early endothelial cells required for the expansion of vascular networks. Together, our findings suggest that migratory events critical to the de novo formation of blood vessels are under the influence of S1P, possibly synthesized via the action of SK2, with signaling mediated by S1P receptors that include S1P(1), S1P(2), and S1P(3).