Betaglycan Is Required for the Establishment of Nephron Endowment in the Mouse

Betaglycan is an accessory receptor for the transforming growth factor-β (TGFβ) superfamily, many members of which play key roles in kidney development. The purpose of this study was to define the role of this co-receptor on fetal murine kidney development. Stereological examination of embryonic and adult betaglycan heterozygous kidneys revealed augmented nephron number relative to littermate controls. Fetal heterozygous kidneys exhibited accelerated ureteric branching, which correlated with augmented nephron development at embryonic day (e) 15.5. In contrast, betaglycan null kidneys exhibited renal hypoplasia from e13.5 and reduced nephron number at e15.5. Quantitative real-time PCR analysis of e11.5–e14.5 kidneys demonstrated that heterozygous kidneys exhibited a transient decrease in Bmp4 expression at e11.5 and a subsequent cascade of changes in the gene regulatory network that governs metanephric development, including significant increases in Pax2, Eya1, Gdnf, Ret, Wnt4, and Wt1 expression. Conversely, gene expression in null kidneys was normal until e13.5, when significant reductions were detected in the expression of Bmp4 as well as other key metanephric regulatory genes. Tgfb1 and Tgfb2 mRNA expression was down-regulated in both nulls and heterozygotes at e13.5 and e14.5. The opposing morphological and molecular phenotypes in betaglycan heterozygote and null mutants demonstrate that the levels of betaglycan must be tightly regulated for optimal kidney development

Specific interaction with the nuclear transporter importin α2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles

Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPα proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMPαs control cellular development, we conducted a yeast two-hybrid screen for IMPα2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPα2 expression coincident with germ-line masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPα2-binding partner. PSPC1-IMPα2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay–based assay demonstrated direct, high-affinity PSPC1 binding to either IMPα2/IMPβ1 or IMPα6/IMPβ1. Coexpression of full-length PSPC1 and IMPα2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMPα2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPα2 isoform or small interfering RNA knockdown of IMPα2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPα2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs

Estrogen-Dependent Gene Expression in the Mouse Ovary

Estrogen (E) plays a pivotal role in regulating the female reproductive system, particularly the ovary. However, the number and type of ovarian genes influenced by estrogen remain to be fully elucidated. In this study, we have utilized wild-type (WT) and aromatase knockout (ArKO; estrogen free) mouse ovaries as an in vivo model to profile estrogen dependent genes. RNA from each individual ovary (n = 3) was analyzed by a microarray-based screen using Illumina Sentrix Mouse WG-6 BeadChip (45,281 transcripts). Comparative analysis (GeneSpring) showed differential expression profiles of 450 genes influenced by E, with 291 genes up-regulated and 159 down-regulated by 2-fold or greater in the ArKO ovary compared to WT. Genes previously reported to be E regulated in ArKO ovaries were confirmed, in addition to novel genes not previously reported to be expressed or regulated by E in the ovary. Of genes involved in 5 diverse functional processes (hormonal processes, reproduction, sex differentiation and determination, apoptosis and cellular processes) 78 had estrogen-responsive elements (ERE). These analyses define the transcriptome regulated by E in the mouse ovary. Further analysis and investigation will increase our knowledge pertaining to how E influences follicular development and other ovarian functions

Expression of TGFβ isoforms in kidneys of betaglycan mutant mice.

<p>Quantitative real time-PCR assessment of (A) <i>Tgfb1</i>; (B) <i>Tgfb2</i>; and (C) <i>Tgfb3</i> using RNA derived from age-matched wildtype (solid bars), betaglycan heterozygous (striped bars), and betaglycan null (clear bars) metanephroi. Values are mean + SEM. n = 4–6 RNA pools/genotype/age. Analysis via one-way ANOVA followed by Tukey's post-hoc analysis conducted separately for each gene at each time point. Significant differences (<i>p</i>&lt;0.05) between genotypes are represented by different letters.</p

Stereological data for betaglycan wildtype, heterozygous and knockout mouse kidneys at e13.5 and e15.5.

<p>Values are mean ± S.D. e13.5: (n)  =  +/+ (5), +/− (9), −/− (6); e15.5: (n)  = 7 mice/genotype. Data were analysed via one-way ANOVA followed by a Tukey's post-hoc analysis. Those groups not sharing a common letter are significantly different (<i>p</i>&lt;0.01).</p

SMAD1 and SMAD3 activation in betaglycan mutant metanephroi.

<p>Quantitative analysis of nuclear pSMAD1 and pSMAD3 expression per microscopic field in wildtype (solid bars), betaglycan heterozygous (striped bars) and betaglycan null (clear bars) metanephroi. n = 4 metanephroi per genotype, with a minimum of 5 fields of view per metanephros. Values are mean + SD. Analysis via one-way ANOVA followed by Tukey's post-hoc analysis. No significant differences were found between genotypes for either pSMAD.</p