Several described growth factors influence the proliferation and regeneration of the intestinal epithelium. Using a transgenic mouse model, we identified a human gene, R-spondin1, with potent and specific proliferative effects on intestinal crypt cells. Human R-spondin1 (hRSpo1) is a thrombospondin domain-containing protein expressed in enteroendocrine cells as well as in epithelial cells in various tissues. Upon injection into mice, the protein induced rapid onset of crypt cell proliferation involving beta-catenin stabilization, possibly by a process that is distinct from the canonical Wnt-mediated signaling pathway. The protein also displayed efficacy in a model of chemotherapy-induced intestinal mucositis and may have therapeutic application in gastrointestinal diseases.

In mammals, female development has traditionally been considered a default process in the absence of the testis-determining gene, Sry. Recently, it has been documented that the gene for R-spondin1 (RSPO1), a novel class of soluble activator for Wnt/beta-catenin signaling, is mutated in two Italian families with female-to-male (XX) sex reversal. To elucidate the role of Rspo1 as a candidate female-determining gene in a mouse model, we generated Rspo1-null (Rspo1(-/-)) mice and found that Rspo1(-/-) XX mice displayed masculinized features including pseudohermaphroditism in genital ducts, depletion of fetal oocytes, male-specific coelomic vessel formation and ectopic testosterone production in the ovaries. Thus, although Rspo1 is required to fully suppress the male differentiation program and to maintain germ cell survival during the development of XX gonads, the loss of its activity has proved to be insufficient to cause complete XX sex reversal in mice. Interestingly, these partial sex-reversed phenotypes of Rspo1(-/-) XX mice recapitulated those of previously described Wnt-4(-/-) XX mice. In accordance with this finding, the expression of Wnt-4 and its downstream genes was deregulated in early Rspo1(-/-) XX gonads, suggesting that Rspo1 may participate in suppressing the male pathway in the absence of Sry and maintaining oocyte survival through positively regulating Wnt-4 signaling.

FGF23 suppresses both serum phosphate and 1,25-dihydroxyvitamin D [1,25D] levels in vivo. Because 1,25D itself is a potent regulator of phosphate metabolism, it has remained unclear whether FGF23-induced changes in phosphate metabolism were caused by a 1,25D-independent mechanism. To address this issue, we intravenously administered recombinant FGF23 to vitamin D receptor (VDR) null (KO) mice as a rapid bolus injection and evaluated the early effects of FGF23. Administration of recombinant FGF23 further decreased the serum phosphate level in VDR KO mice, accompanied by a reduction in renal sodium-phosphate cotransporter type IIa (NaPi2a) protein abundance and a reduced renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alphaOHase) mRNA level. Thus FGF23-induced changes in NaPi2a and 1alphaOHase expression are independent of the 1,25D/VDR system. However, 24-hydroxylase (24OHase) mRNA expression remained undetectable by the treatment with FGF23. We also analyzed the regulatory mechanism for FGF23 expression. The serum FGF23 level was almost undetectable in VDR KO mice, whereas dietary calcium supplementation significantly increased circulatory levels of FGF23 and its mRNA abundance in bone. This finding indicates that calcium is another determinant of FGF23 production that occurs independently of the VDR-mediated mechanism. In contrast, dietary phosphate supplementation failed to induce FGF23 expression in the absence of VDR, whereas marked elevation in circulatory FGF23 was observed in wild-type mice fed with a high-phosphate diet. Taken together, FGF23 works, at least in part, in a VDR-independent manner, and FGF23 production is also regulated by multiple mechanisms involving VDR-independent pathways.

The R-spondin (Rspo) protein family is a recently described group of four distinct human secreted proteins. Reported activities for Rspo proteins include essential roles in vertebrate development and their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both Rspo and canonical Wnt signaling result in the activation of beta-catenin. In a general functional screen for human secreted proteins using transgenic mouse models, we identified human R-spondin1 (hRspo1) protein as a potent and specific mitogen for the gastrointestinal epithelium and demonstrated a potential therapeutic application for the protein in mouse models of cancer therapy-induced mucositis. In contrast to previous studies, our data indicated only partial overlap between Wnt and Rspo ligand activities, suggesting that there may be independent receptor/signaling pathways for Rspo proteins that intersect those of Wnt at the level of beta-catenin. Here we summarize the current reported data on the Rspo family and discuss these results in terms of alternate mechanisms of action. We have extended our observations on the potential therapeutic application of Rspo proteins by showing that all four human Rspo family members are capable of inducing epithelial proliferation and report the first non-vertebrate Rspo family member.

Mutations in the connexin 26 gene (GJB2), which encodes a gap-junction protein and is expressed in the inner ear, have been shown to be responsible for a major part of nonsyndromic hereditary prelingual (early-childhood) deafness in Caucasians. We have sequenced the GJB2 gene in 39 Japanese patients with prelingual deafness (group 1), 39 Japanese patients with postlingual progressive sensorineural hearing loss (group 2), and 63 Japanese individuals with normal hearing (group 3). Three novel mutations were identified in group 1: a single nucleotide deletion (235delC), a 16-bp deletion (176-191 del (16)), and a nonsense mutation (Y136X) in five unrelated patients. The 235delC mutation was most frequently observed, accounting for seven alleles in 10 mutant alleles. Screening of 203 unrelated normal individuals for the three mutations indicated that the carrier frequency of the 235delC mutation was 2/203 in the Japanese population. No mutation was found in group-2 patients. We also identified two novel polymorphisms (E114G and I203T) as well as two previously reported polymorphisms (V27I andV37I). Genotyping with these four polymorphisms allowed normal Japanese alleles to be classified into seven haplotypes. All 235delC mutant alleles identified in four patients resided only on haplotype type 1. These findings indicate that GJB2 mutations are also responsible for prelingual deafness in Japan.

DFN3, an X chromosome-linked nonsyndromic mixed deafness, is caused by mutations in the BRN-4 gene, which encodes a POU transcription factor. Brn-4-deficient mice were created and found to exhibit profound deafness. No gross morphological changes were observed in the conductive ossicles or cochlea, although there was a dramatic reduction in endocochlear potential. Electron microscopy revealed severe ultrastructural alterations in cochlear spiral ligament fibrocytes. The findings suggest that these fibrocytes, which are mesenchymal in origin and for which a role in potassium ion homeostasis has been postulated, may play a critical role in auditory function.

Abstract. Tau proteins are a class of low molecular mass microtubule-associated proteins that are specifically expressed in the nervous system. A cDNA clone of adult rat tau was isolated and sequenced. To analyze functions of tau proteins in vivo, we carried out transfection experiments. A fibroblast cell line, which was transfected with the cDNA, expressed three bands of tau, while six bands were expressed in rat brain. After dephosphorylation, one of the three bands disappeared, demonstrating directly that phosphorylation was involved in the multiplicity of tau. Morphologically, we observed a thick bundle formation of microtubules in the transiently and stably tau-genetransfected cells. In addition, we found that the production of tubulin was prominently enhanced in the stably transfected cells. Thus, we suppose that tau proteins promote polymerization of tubulin, form bundles of microtubules in vivo, and play important roles in growing and maintaining nerve cell processes.

R-spondin1 stimulates the proliferation of intestinal stem cells through the Wnt signaling pathway and protects against graft-versus-host disease.