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

Heart and Liver Defects and Reduced Transforming Growth Factor β2 Sensitivity in Transforming Growth Factor β Type III Receptor-Deficient Embryos

The type III transforming growth factor β (TGFβ) receptor (TβRIII) binds both TGFβ and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of TβRIII signaling in vivo is not known. In this study, we have sought to determine the role of TβRIII during development. We identified the predominant expression sites of ΤβRIII mRNA as liver and heart during midgestation and have disrupted the murine TβRIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in ΤβRIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that TβRIII is required during murine somatic development. To assess the effects of the absence of TβRIII on the function of its ligands, primary fibroblasts were generated from TβRIII-null and wild-type embryos. Our results indicate that TβRIII deficiency differentially affects the activities of TGFβ ligands. Notably, TβRIII-null cells exhibited significantly reduced sensitivity to TGFβ2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that TβRIII is an important modulator of TGFβ2 function in embryonic fibroblasts and that reduced sensitivity to TGFβ2 may underlie aspects of the TβRIII mutant phenotype

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

Betaglycan mutant renal mRNA expression.

<p>Quantitative real time PCR assessment of genes involved in metanephric development 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

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

Stereological analysis of female wildtype (+/+) and betaglycan heterozygous (+/−) mouse kidneys at postnatal day 30.

<p>Values are mean ± S.D. (n)  = 7 mice/genotype,</p><p>*<i>p</i>&lt;0.05,</p><p>**<i>p</i>&lt;0.0001.</p

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