47Pulmonary endothelial cells (ECs) are an essential component of the gas exchange 48 machinery of the lung alveolus. Despite this, the extent and function of lung EC heterogeneity 49 remains incompletely understood. Using single-cell analytics, we identify multiple EC 50 populations in the mouse lung, including macrovascular endothelium (maEC), microvascular 51 endothelium (miECs), and a new population we have termed Car4-high ECs. Car4-high ECs 52 express a unique gene signature, and ligand-receptor analysis indicates they are primed to 53 receive reparative signals from alveolar type I cells. After acute lung injury, they are 54 preferentially localized in regenerating regions of the alveolus. Influenza infection reveals the 55 emergence of a population of highly proliferative ECs that likely arise from multiple miEC 56 populations and contribute to alveolar revascularization after injury. These studies map EC 57 heterogeneity in the adult lung and characterize the response of novel EC subpopulations 58 required for tissue regeneration after acute lung injury. 59 Significance 60Using transcriptional profiling of the pulmonary vascular endothelium and confirmation at 61 the RNA and protein levels, we have revealed extensive EC heterogeneity throughout the 62 vasculature of the lungs. We show that a subpopulation of endothelium re-enters the cell cycle 63 and proliferates in response to acute injury, whereas another subpopulation is enriched in 64 vasculogenic gene expression. These data provide foundational information regarding the 65 biological complexity of lung ECs, which will contribute to the development of novel tools to 66 enhance regeneration of the lung following injury. 67 whether such EC subpopulations contribute to in vivo tissue homeostasis and response to injury 93 in the adult lung remains unknown. 94To address these questions and to define pulmonary EC heterogeneity at homeostasis 95 and during regeneration, we utilized single cell RNA sequencing (scRNA-seq) analysis of the 96 adult mouse lung, both uninjured and after acute influenza-induced viral injury. In addition to 97 identifying microvascular (miEC) and both arterial and venous macrovascular (maEC) 98 populations, we identified a new population we have termed Car4-high ECs that possess a 99 unique transcriptome. Car4-high ECs express high levels of Car4 and Cd34, are found 100 throughout the lung periphery at homeostasis, and are primed to respond to Vegfa signaling 101 based on their high expression of Vegf receptors, which corresponds to a receptor-ligand 102 interaction analysis between Car4-high ECs and AT1 cells, their epithelial co-partners in gas 103 exchange. Car4-high ECs are enriched in the regenerating zones surrounding the most 104 damaged regions of the lung following influenza-or bleomycin-induced lung injury during the 105 subsequent tissue regeneration process. Influenza injury revealed the emergence of a unique 106 population of highly proliferative ECs, which are closely related to Car4-low miECs in gene 107 expression ...
The lung alveolus is the functional unit of the respiratory system required for gas exchange. During the transition to air breathing at birth, biophysical forces are thought to shape the emerging tissue niche. However, the intercellular signaling that drives these processes remains poorly understood. Applying a multimodal approach, we identified alveolar type 1 (AT1) epithelial cells as a distinct signaling hub. Lineage tracing demonstrates that AT1 progenitors align with receptive, force-exerting myofibroblasts in a spatial and temporal manner. Through single-cell chromatin accessibility and pathway expression (SCAPE) analysis, we demonstrate that AT1-restricted ligands are required for myofibroblasts and alveolar formation. These studies show that the alignment of cell fates, mediated by biophysical and AT1-derived paracrine signals, drives the extensive tissue remodeling required for postnatal respiration.
Eph/ephrins drive cell segregation and boundary formation. O’Neill et al. discover that segregation is driven by unidirectional kinase-dependent EphB signaling. Unidirectional signaling generates a cortical actin differential between ephrin-B1– and EphB2-expressing cells and requires ROCK activity for cell segregation.
GNE myopathy, previously termed hereditary inclusion body myopathy (HIBM), is an adult-onset neuromuscular disorder characterized by progressive muscle weakness. The disorder results from biallelic mutations in GNE, encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid synthesis. GNE myopathy, associated with impaired glycan sialylation, has no approved therapy. Here we test potential sialylation-increasing monosaccharides for their effectiveness in prophylaxis (at the embryonic and neonatal stages) and therapy (after the onset of symptoms) by evaluating renal and muscle hyposialylation in a knock-in mouse model (Gne p.M712T) of GNE myopathy. We demonstrate that oral mannosamine (ManN), but not sialic acid (Neu5Ac), mannose (Man), galactose (Gal), or glucosamine (GlcN), administered to pregnant female mice has a similar prophylactic effect on renal hyposialylation, pathology and neonatal survival of mutant offspring, as previously shown for N-acetylmannosamine (ManNAc) therapy. ManN may be converted to ManNAc by a direct, yet unknown, pathway, or may act through another mode of action. The other sugars (Man, Gal, GlcN) may either not cross the placental barrier (Neu5Ac) and/or may be able to directly increase sialylation. Because GNE myopathy patients will likely require treatment in adulthood after onset of symptoms, we also administered ManNAc (1 or 2 g/kg/day for 12 weeks), Neu5Ac (2g/kg/day for 12 weeks), or ManN (2g/kg/day for 6 weeks) in drinking water to 6 month old mutant Gne p.M712T mice. All three therapies markedly improved the muscle and renal hyposialylation, as evidenced by lectin histochemistry for overall sialylation status and immunoblotting of specific sialoproteins. These preclinical data strongly support further evaluation of oral ManNAc, Neu5Ac and ManN as therapy for GNE myopathy and conceivably for certain glomerular diseases with hyposialylation.
Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder characterized by craniofacial, skeletal, and neurological anomalies and is caused by mutations in EFNB1. Heterozygous females are more severely affected by CFNS than hemizygous males, a phenomenon called cellular interference that results from EPHRIN-B1 mosaicism. In Efnb1 heterozygous mice, mosaicism for EPHRIN-B1 results in cell sorting and more severe phenotypes than Efnb1 hemizygous males, but how craniofacial dysmorphology arises from cell segregation is unknown and CFNS etiology therefore remains poorly understood. Here, we couple geometric morphometric techniques with temporal and spatial interrogation of embryonic cell segregation in mouse mutant models to elucidate mechanisms underlying CFNS pathogenesis. By generating EPHRIN-B1 mosaicism at different developmental timepoints and in specific cell populations, we find that EPHRIN-B1 regulates cell segregation independently in early neural development and later in craniofacial development, correlating with the emergence of quantitative differences in face shape. Whereas specific craniofacial shape changes are qualitatively similar in Efnb1 heterozygous and hemizygous mutant embryos, heterozygous embryos are quantitatively more severely affected, indicating that Efnb1 mosaicism exacerbates loss of function phenotypes rather than having a neomorphic effect. Notably, neural tissue-specific disruption of Efnb1 does not appear to contribute to CFNS craniofacial dysmorphology, but its disruption within neural crest cell-derived mesenchyme results in phenotypes very similar to widespread loss. EPHRIN-B1 can bind and signal with EPHB1, EPHB2, and EPHB3 receptor tyrosine kinases, but the signaling partner(s) relevant to CFNS are unknown. Geometric morphometric analysis of an allelic series of Ephb1; Ephb2; Ephb3 mutant embryos indicates that EPHB2 and EPHB3 are key receptors mediating Efnb1 hemizygous-like phenotypes, but the complete loss of EPHB1-3 does not fully
Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.
In vertebrates, the Eph/ephrin family of signaling molecules is a large group of membrane-bound proteins that signal through a myriad of mechanisms and effectors to play diverse roles in almost every tissue and organ system. Though Eph/ephrin signaling has functions in diverse biological processes, one core developmental function is in the regulation of cell position and tissue morphology by regulating cell migration and guidance, cell segregation, and boundary formation. Often, the role of Eph/ephrin signaling is to translate patterning information into physical movement of cells and changes in morphology that define tissue and organ systems. In this review, we focus on recent advances in the regulation of these processes, and our evolving understanding of the in vivo signaling mechanisms utilized in distinct developmental contexts.
C-O activation of mesylates by a palladium catalyst and subsequent cross-coupling with potassium cyclopropyltrifluoroborate have been achieved with high yield. Both electron-enriched and electron-deficient aryl mesylates are suitable electrophilic partners for the Suzuki-Miyaura reaction. The scope was successfully extended to heteroaryl mesylates with yields up to 94%.
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