Estrogens regulate early embryonic development of the olfactory sensory system via estrogen-responsive glia

ABSTRACT Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour.

The Atypical Cyclin-Dependent Kinase 5 (Cdk5) Guards Podocytes from Apoptosis in Glomerular Disease While Being Dispensable for Podocyte Development

Cyclin-dependent kinase 5 (Cdk5) is expressed in terminally differentiated cells, where it drives development, morphogenesis, and survival. Temporal and spatial kinase activity is regulated by specific activators of Cdk5, dependent on the cell type and environmental factors. In the kidney, Cdk5 is exclusively expressed in terminally differentiated glomerular epithelial cells called podocytes. In glomerular disease, signaling mechanisms via Cdk5 have been addressed by single or combined conventional knockout of known specific activators of Cdk5. A protective, anti-apoptotic role has been ascribed to Cdk5 but not a developmental phenotype, as in terminally differentiated neurons. The effector kinase itself has never been addressed in animal models of glomerular disease. In the present study, conditional and inducible knockout models of Cdk5 were analyzed to investigate the role of Cdk5 in podocyte development and glomerular disease. While mice with podocyte-specific knockout of Cdk5 had no developmental defects and regular lifespan, loss of Cdk5 in podocytes increased susceptibility to glomerular damage in the nephrotoxic nephritis model. Glomerular damage was associated with reduced anti-apoptotic signals in Cdk5-deficient mice. In summary, Cdk5 acts primarily as master regulator of podocyte survival during glomerular disease and—in contrast to neurons—does not impact on glomerular development or maintenance.

Estrogen regulates early embryonic development of the olfactory sensory system via estrogen responsive glia

Estrogen is well-known to regulate development of sexual dimorphisms of the brain, however its role in the brain during early embryonic development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs specifically in a type of glia located within the OB, which we name estrogen-responsive olfactory bulb/EROB cells. With estrogen activity, EROB cells extend their ramified projections that overlay the OB outermost layer and tightly interact with olfactory sensory neurons (OSNs) at the olfactory glomeruli. Pharmacologically inhibiting estrogen activity and/or EROB cell ablation impedes olfactory glomerular development, including OSN pathfinding, topological organisation of olfactory glomeruli and inhibitory neurogenesis in the OB. Furthermore, activation of this estrogen/EROB-dependent mechanism decreases the intrinsic neuronal activity primarily in the OB, and this alteration of estrogen signalling disrupts olfaction-mediated behaviour. We propose that estrogen acts on glia to regulate development of functional OB circuits, thereby modulating the local intrinsic excitability in the OB and olfaction-mediated behaviour. Our data also suggest a possibility that the estrogen/EROB cascade may be an important site of action for environmental estrogens causative of neurodevelopmental impairments in animals and humans.

Early B Cell Factor 1 (EBF1) Regulates Glomerular Development by Controlling Mesangial Maturation and Consequently COX-2 Expression

BackgroundWe recently showed the transcription factor Early B cell factor 1 (EBF1) is essential for the last stages of metanephric development, and that mice globally deficient in EBF1 display impaired maturation of peripheral glomeruli. EBF1 is present within multiple glomerular cell types, including the glomerular mesangium and podocytes.MethodsTo identify which cell type is driving the glomerular developmental defects in the global EBF1 knockout mice, we deleted EBF1 from the mesangium/pericytes (Foxd1-cre) or podocytes (Podocin-cre) in mice.ResultsDeletion of EBF1 from Foxd1 lineage cells resulted in hypoplastic kidneys, poorly differentiated peripheral glomeruli, and decreased proximal tubular mass in the outer cortex. Renal insufficiency was apparent at P21 when proteinuria presents, fibrosis of both the glomeruli and interstitium rapidly progresses, microthrombi appear, and hematuria develops. Approximately half of the Foxd1+, Ebf1fl/fl mice die before they are 3 months old. Mice with podocyte-targeted deletion of EBF1 exhibited no developmental abnormalities. Mice with Ebf1 deficiency in Foxd1 lineage cells shared characteristics with Ptgs2/COX-2–insufficient models, and mechanistic investigation revealed impaired calcineurin/NFATc1 activation and decreased COX-2 expression. Deletion of COX-2 from the interstitial/mesangial lineage displayed a less severe phenotype than EBF1 deficiency in mice. Overexpressing COX-2 in the EBF1-deficient mice, however, partially restored glomerular development.ConclusionsThe results suggest that EBF1 regulates metanephric development at the last stages of glomerular maturation through its actions in the stromal progenitor (Foxd1+) lineage where it mediates proper regulation of calcineurin/NFAT signaling and COX-2 expression.

A Novel Role for GATA3 in Mesangial Cells in Glomerular Development and Injury

BackgroundGATA3 is a dual-zinc finger transcription factor that regulates gene expression in many developing tissues. In the kidney, GATA3 is essential for ureteric bud branching, and mice without it fail to develop kidneys. In humans, autosomal dominant GATA3 mutations can cause renal aplasia as part of the hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that includes mesangioproliferative GN. This suggests that GATA3 may have a previously unrecognized role in glomerular development or injury.MethodsTo determine GATA3’s role in glomerular development or injury, we assessed GATA3 expression in developing and mature kidneys from Gata3 heterozygous (+/−) knockout mice, as well as injured human and rodent kidneys.ResultsWe show that GATA3 is expressed by FOXD1 lineage stromal progenitor cells, and a subset of these cells mature into mesangial cells (MCs) that continue to express GATA3 in adult kidneys. In mice, we uncover that GATA3 is essential for normal glomerular development, and mice with haploinsufficiency of Gata3 have too few MC precursors and glomerular abnormalities. Expression of GATA3 is maintained in MCs of adult kidneys and is markedly increased in rodent models of mesangioproliferative GN and in IgA nephropathy, suggesting that GATA3 plays a critical role in the maintenance of glomerular homeostasis.ConclusionsThese results provide new insights on the role GATA3 plays in MC development and response to injury. It also shows that GATA3 may be a novel and robust nuclear marker for identifying MCs in tissue sections.

MAGI-2 and scaffold proteins in glomerulopathy

In many cells and tissues, including the glomerular filtration barrier, scaffold proteins are critical in optimizing signal transduction by enhancing structural stability and functionality of their ligands. Recently, mutations in scaffold protein membrane-associated guanylate kinase inverted 2 (MAGI-2) encoding gene were identified among the etiology of steroid-resistant nephrotic syndrome. MAGI-2 interacts with core proteins of multiple pathways, such as transforming growth factor-β signaling, planar cell polarity pathway, and Wnt/β-catenin signaling in podocyte and slit diaphragm. Through the interaction with its ligand, MAGI-2 modulates the regulation of apoptosis, cytoskeletal reorganization, and glomerular development. This review aims to summarize recent findings on the role of MAGI-2 and some other scaffold proteins, such as nephrin and synaptopodin, in the underlying mechanisms of glomerulopathy.

SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects Albuminuria in Allografts

BackgroundWe previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin–binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development.MethodsWe used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli.ResultsExpression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3–binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration.ConclusionsWe demonstrate a novel mechanism that may explain SHROOM3’s dichotomous associations in glomerular versus nonglomerular compartments in CKD

Nucleoporin 133 deficiency leads to glomerular damage in zebrafish

Although structural nuclear pore proteins (nucleoporins) are seemingly required in every cell type to assemble a functional nuclear transport machinery, mutations or deregulation of a subset of them have been associated with specific human hereditary diseases. In particular, previous genetic studies of patients with nephrotic syndrome identified mutations inNup107that impaired the expression or the localization of its direct partner at nuclear pores, Nup133. In the present study, we characterized the zebrafishnup133orthologous gene and its expression pattern during larval development. Morpholino-mediated gene knockdown revealed that Nup133 depletion in zebrafish larvae leads to the formation of kidney cysts, a phenotype that can be rescued by co-injection of wild type mRNA. Analysis of different markers for tubular and glomerular development shows that the overall kidney development is not affected bynup133knockdown. On the other hand, we demonstrate thatnup133is essential for the organization and functional integrity of the pronephric glomerular filtration barrier, as its downregulation results in proteinuria and moderate foot process effacement, mimicking some of the abnormalities typically featured by patients with nephrotic syndrome. These data indicate thatnup133is a new gene required for proper glomerular structure and function in zebrafish.