The small RNA expression profile of the developing murine urinary and reproductive systems

AbstractmicroRNAs (miRNAs) are small non-coding RNAs with fundamental roles in the regulation of gene expression. miRNAs assemble with Argonaute (Ago) proteins to miRNA-protein complexes (miRNPs), which interact with distinct binding sites on mRNAs and regulate gene expression. Specific miRNAs are key regulators of tissue and organ development and it has been shown in mammals that miRNAs are also involved in the pathogenesis of many diseases including cancer. Here, we have characterized the miRNA expression profile of the developing murine genitourinary system. Using a computational approach, we have identified several miRNAs that are specific for the analyzed tissues or the developmental stage. Our comprehensive miRNA expression atlas of the developing genitourinary system forms an invaluable basis for further functional in vivo studies

Lymphotoxin β receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE−/− mice

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE−/− mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin β receptor (LTβR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE−/− mice with LTβR-Ig to interrupt LTβR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTβR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall

Wilms' Tumor Protein Wt1 Is an Activator of the Anti-Müllerian Hormone Receptor Gene Amhr2

The Wilms' tumor protein Wt1 plays an essential role in mammalian urogenital development. WT1 mutations in humans lead to a variety of disorders, including Wilms' tumor, a pediatric kidney cancer, as well as Frasier and Denys-Drash syndromes. Phenotypic anomalies in Denys-Drash syndrome include pseudohermaphroditism and sex reversal in extreme cases. We have used cDNA microarray analyses on Wt1 knockout mice to identify Wt1-dependent genes involved in sexual development. The gene most dramatically affected by Wt1 inactivation was Amhr2, encoding the anti-Müllerian hormone (Amh) receptor 2. Amhr2 is an essential factor for the regression of the Müllerian duct in males, and mutations in AMHR2 lead to the persistent Müllerian duct syndrome, a rare form of male pseudohermaphroditism. Here we show that Wt1 and Amhr2 are coexpressed during urogenital development and that the Wt1 protein binds to the promoter region of the Amhr2 gene. Inactivation and overexpression of Wt1 in cell lines was followed by immediate changes of Amhr2 expression. The identification of Amhr2 as a Wt1 target provides new insights into the role of Wt1 in sexual differentiation and indicates, in addition to its function in early gonad development and sex determination, a novel function for Wt1, namely, in Müllerian duct regression

BOR-Syndrome-Associated Eya1 Mutations Lead to Enhanced Proteasomal Degradation of Eya1 Protein

Mutations in the human EYA1 gene have been associated with several human diseases including branchio-oto (BO) and branchio-oto-renal (BOR) syndrome, as well as congenital cataracts and ocular anterior segment anomalies. BOR patients suffer from severe malformations of the ears, branchial arches and kidneys. The phenotype of Eya1-heterozygous mice resembles the symptoms of human patients suffering from BOR syndrome. The Eya1 gene encodes a multifunctional protein that acts as a protein tyrosine phosphatase and a transcriptional coactivator. It has been shown that Eya1 interacts with Six transcription factors, which are also required for nuclear translocation of the Eya1 protein. We investigated the effects of seven disease-causing Eya1 missense mutations on Eya1 protein function, in particular cellular localization, ability to interact with Six proteins, and protein stability. We show here that the BOR-associated Eya1 missense mutations S454P, L472R, and L550P lead to enhanced proteasomal degradation of the Eya1 protein in mammalian cells. Moreover, Six proteins lead to a significant stabilization of Eya1, which is caused by Six-mediated protection from proteasomal degradation. In case of the mutant L550P, loss of interaction with Six proteins leads to rapid protein degradation. Our observations suggest that protein destabilization constitutes a novel disease causing mechanism for Eya1

Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development▿

The eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies. For a better understanding of Eya1's function, we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as novel interaction partners of Eya1. We confirmed the interactions by glutathione S-transferase (GST) pulldown analysis and coimmunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Using reverse transcriptase PCR (RT-PCR) and in situ hybridization, we show that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. By morpholino-mediated knockdown, we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 ortholog as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype, with characteristic defects in ear and branchial arch formation

The BOR mutant L550P fails to translocate into the nucleus in presence of Six.

<p>(A) Eya domain of human Eya1 depicting the disease associated missense mutations included in this study. BOR branchio-oto-renal; BO branchio-otic; OD ocular defects. (B) Cellular localization of Eya1 and disease-associated Eya1 mutants. COS-7 cells were transfected with expression plasmids encoding EGFP-Eya1 or mutants either alone or in combination with Six2. Insets show counterstaining with Hoechst dye. (C) COS-7 cells were transfected with HA-tagged <i>Eya1</i> or mutants together with FLAG-Six2 or empty vector. Nuclear (N) and cytoplasmic (C) extracts were analyzed by immunoblotting.</p

Eya1 degradation occurs via the proteasome and is inhibited by Six proteins.

<p>(A) Eya1 and its mutants accumulate in the presence of the proteasome inhibitor lactacystin. COS-7 cells were transfected with wild type or mutant HA-Eya1 or empty vector. 24 h post-transfection cells were treated with lactacystin (1 µM). Cells were lysed and Eya1 was detected by immunoblotting. (B) Eya1 is ubiquitinated. COS-7 cells were co-transfected with HA-Eya1 and HA-ubiquitin or His-ubiquitin. 24 h later, cells were treated with MG132 (10 µM) or DMSO for 24 h. For <i>in vivo</i> ubiquitination assay, His-ubiquitin-labelled proteins were purified from cell lysates followed by immunoblotting and detection of Eya1 using anti-Eya1 antibody. Asterisks indicate unspecific signals. (C) Schematic representation of the Eya1 mutants used for <i>in vivo</i> ubiquitination assay. Position of lysine residues is indicated in the scheme of full-length Eya1. (D) Eya1 ubiquitination occurs in two distinct regions of the Eya domain. COS-7 cells were co-transfected with His-ubiquitin and the indicated <i>Eya1</i> expression constructs, treated with MG132 (10 µM), and subjected to <i>in vivo</i> ubiquitination assay followed by immunoblotting and detection of Eya1 using anti-HA antibody. (E) Six proteins inhibit ubiquitination of Eya1. COS-7 cells overexpressing wild type or mutant HA-Eya1, His-ubiquitin and Six2 or empty vector were subjected to <i>in vivo</i> ubiquitination assay. Ubiquitinated Eya1 protein was detected by immunoblotting using anti-HA antibody. β-actin was used as a loading control for total cell lysate.</p

Six proteins stabilize Eya1 and Eya1 mutants with the exception of L550P.

<p>(A) COS-7 cells were transfected with empty vector (control), HA-Eya1 or mutants, and FLAG-Six2. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting using anti-HA and anti-FLAG antibody. (B) Endogenous Eya1 is stabilized in presence of Six2. MK4 cells were transfected with <i>Eya1</i>-specific or non-target control siRNA and Eya1 protein levels were detected by immunoblotting using anti-Eya1 antibody (left). MK4 cells stably expressing FLAG-Six2 were subject to immunoblot analyses using Eya1-specific antibody (right). Asterisk indicates unspecific signal. (C) Six induces accumulation of Eya1 protein in different cell lines via the conserved Six and homeo domain. COS-7 and U2OS cells were transfected with HA-<i>Eya1</i> and expression vectors for <i>Six1</i>, <i>Six2</i>, C-terminal domain of Six2 (CD), <i>GDNF</i> and <i>H-Ras</i>. Eya1 protein levels were detected by immunoblotting using anti-HA antibody. (D) Schematic representation of the Six2 deletion constructs used for characterization of Eya1 stabilization (left). COS-7 cells were co-transfected with HA-Eya1, Six2 or deletion constructs as indicated. Eya1 protein levels were analyzed by immunoblotting.</p

Eya1 stabilization requires interaction with Six proteins.

<p>COS-7 cells were transfected with HA-Eya1 or mutants and FLAG-Six2 as indicated (left). In case of the mutant L550P cells were treated with MG132 (10 µM) to enhance protein levels (right). Six2 was immunoprecipitated using anti-FLAG antibody, and interacting Eya1 was detected by immunoblotting.</p