Involvement of GLCCI1 in mouse spermatogenesis.

Spermatid production is a complex regulatory process in which coordination between hormonal control and apoptosis plays a pivotal role in maintaining a balanced number of sperm cells. Apoptosis in spermatogenesis is controlled by pro-apoptotic and anti-apoptotic molecules. Hormones involved in the apoptotic process during spermatogenesis include gonadotrophins, sex hormones, and glucocorticoid (GC). GC acts broadly as an apoptosis inducer by binding to its receptor (glucocorticoid receptor: GR) during organ development processes, such as spermatogenesis. However, the downstream pathway induced in GC-GR signaling and the apoptotic process during spermatogenesis remains poorly understood. We reported previously that GC induces full-length glucocorticoid-induced transcript 1 (GLCCI1-long), which functions as an anti-apoptotic mediator in thymic T cell development. Here, we demonstrate that mature murine testis expresses a novel isoform of GLCCI1 protein (GLCCI1-short) in addition to GLCCI1-long. We demonstrate that GLCCI1-long is expressed in spermatocytes along with GR. In contrast, GLCCI1-short is primarily expressed in spermatids where GR is absent; instead, the estrogen receptor is expressed. GLCCI1-short also binds to LC8, which is a known mediator of the anti-apoptotic effect of GLCCI1-long. A luciferase reporter assay revealed that ß-estradiol treatment synergistically increased Glcci1-short promotor-driven luciferase activity in Erα-overexpressing cells. Together with the evidence that the conversion of testosterone to estrogen is preceded by aromatase expression in spermatids, we hypothesize that estrogen induces GLCCI1-short, which, in turn, may function as a novel anti-apoptotic mediator in mature murine testis.

Wolf-Hirschhorn syndrome candidate 1-like 1 epigenetically regulates nephrin gene expression.

Altered expression of nephrin underlies the pathophysiology of proteinuria in both congenital and acquired nephrotic syndrome. However, the epigenetic mechanisms of nephrin gene regulation remain elusive. Here, we show that Wolf-Hirschhorn syndrome candidate 1-like 1 long form (WHSC1L1-L) is a novel epigenetic modifier of nephrin gene regulation. WHSC1L1-L was associated with histone H3K4 and H3K36 in human embryonic kidney cells. WHSC1L1-L gene was expressed in the podocytes, and functional protein product was detected in these cells. WHSC1L1-L was found to bind nephrin but not other podocyte-specific gene promoters, leading to its inhibition/suppression, abrogating the stimulatory effect of WT1 and NF-κB. Gene knockdown of WHSC1L1-L in primary cultured podocytes accelerated the transcription of nephrin but not CD2AP. An in vivo zebrafish study involving the injection of Whsc1l1 mRNA into embryos demonstrated an apparent reduction of nephrin mRNA but not podocin and CD2AP mRNA. Immunohistochemistry showed that both WHSC1L1-L and nephrin emerged at the S-shaped body stage in glomeruli. Immunofluorescence and confocal microscopy displayed WHSC1L1 to colocalize with trimethylated H3K4 in the glomerular podocytes. Chromatin immunoprecipitation assay revealed the reduction of the association of trimethylated H3K4 at the nephrin promoter regions. Finally, nephrin mRNA was upregulated in the glomerulus at the early proteinuric stage of mouse nephrosis, which was associated with the reduction of WHSC1L1. In conclusion, our results demonstrate that WHSC1L1-L acts as a histone methyltransferase in podocytes and regulates nephrin gene expression, which may in turn contribute to the integrity of the slit diaphragm of the glomerular filtration barrier.

USP40 gene knockdown disrupts glomerular permeability in zebrafish.

Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.

Proper Notch activity is necessary for the establishment of proximal cells and differentiation of intermediate, distal, and connecting tubule in Xenopus pronephros development.

BACKGROUND: Notch signaling in pronephros development has been shown to regulate establishment of glomus and proximal tubule, but how Notch signal works on competency of pronephric anlagen during the generation of pronephric components remains to be understood. RESULTS: We investigated how components of pronephros (glomus, proximal tubule, intermediate tubule, distal tubule, and connecting tubule) were generated in Xenopus embryos by timed overactivation and suppression of Notch signaling. Notch activation resulted in expansion of the glomus and disruption of the proximal tubule formation. Inhibition of Notch signaling reduced expression of wt1 and XSMP-30. In addition, when Notch signaling was overactivated at stage 20 on, intermediate, distal, and connecting tubule markers, gremlin and clcnkb, were decreased while Notch down-regulation increased gremlin and clcnkb. Similar changes were observed with segmental markers, cldn19, cldn14, and rhcg on activation or inhibition of Notch. Although Notch did not affect the expression of pan-pronephric progenitor marker, pax2, its activation inhibited lumen formation in the pronephros. CONCLUSIONS: Notch signal is essential for glomus and proximal tubule development and inhibition of Notch is critical for the differentiation of the intermediate, distal, and connecting tubule.

Human sodium phosphate transporter 4 (hNPT4/SLC17A3) as a common renal secretory pathway for drugs and urate.

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Transcriptional regulatory elements of hif1α in a distal locus of islet1 in Xenopus laevis.

Adult mammalian hearts are not regenerative. However, recent studies have evidenced that hypoxia enhances their regeneration. Islet1 (isl1) is known as a cardiac progenitor marker, which is quiescent in adult mammal hearts. In Xenopus hearts, transcriptional activation of isl1 was shown during cardiac regeneration of froglets at 3 months after metamorphosis. In this study, we examined transcriptional regulation of isl1 focusing on hypoxia-inducible factor 1α (hif1α) in Xenopus heart. We found that hif1α expression was increased in response to cardiac injury and overexpression of hif1α upregulated mRNA expression of isl1. Multiple conservation analysis including 9 species revealed that 8 multiple conserved regions (MCRs) were present upstream of isl1. DNA sequence analysis using JASPAR showed hif1α binding motifs in MCRs. By luciferase reporter assay and chromatin immunoprecipitation analysis, we found that hif1α directly bound to hif1α motifs in the most distant MCR8 and showed a specific transcriptional activity on the MCR8. In the luciferase assay using constructs carrying MCR8 without a responsive motif of hif1α, the reporter activity was lost. Pharmacologically inhibition of hif1α affected isl1 transcription and downstream events including cardiac phenotypes, suggesting functional defects of islet1. Contrarily in murine hearts, transcription of isl1 was unresponsive even after cryoinjury to adult hearts while hif1α mRNA was induced. In comparative analysis of multiple alignment, hif1α elements present in MCR8 of Xenopus or zebrafish were found to be disrupted as species are evolutionarily distant from Xenopus and zebrafish. Our results suggested an altered switch of isl1 transcription between mammals and Xenopus laevis.

Xenopus slc7a5 is essential for notochord function and eye development.

slc7a5 (also known as LAT1), largely accepted as an amino acid transporter, has been shown to play important roles in cancer and developmental processes. Because knockout mice of Slc7a5 are embryonically lethal due to placental defects, it is difficult to evaluate its role in early development. In this study, expression and function of slc7a5 were evaluated in Xenopus laevis embryos that develop without a placenta. Expression of slc7a5 was detected in the notochord and in the eye and it was not co-localized with slc3a2, which helps slc7a5 to localize at the plasma membrane, before the late neurula stage. Loss-of-function experiment with a morpholino antisense oligonucleotide led to defect of neural and non-neural patterning, inhibition of primary neurogenesis, and disruption of eye development. Disruption of neural development and primary neurogenesis was likely due to impaired notochord development as sonic hedgehog (shh) signaling pathway was compromised in slc7a5-inhibited embryos. These results suggest that slc7a5 is required for notochord development and subsequent primary neurogenesis via shh/gli signaling and for eye development. These novel developmental roles of slc7a5 appeared to be independent of transport function at least before the late neurula stage.

XSu(H)2 is an essential factor for gene expression and morphogenesis of the Xenopus gastrula embryo.

The CSL (CBF-1, Suppressor of Hairless, Lag-1) transcriptional factor is an important mediator of Notch signal transduction. It plays a key role in cell fate determination by cell-cell interaction. CSL functions as a transcriptional repressor before the activation of Notch signaling. However, once Notch signaling is activated, CSL is converted into a transcriptional activator. It remains unclear if CSL has any function during early development before neurogenesis, while transcriptional products exist from the maternal stage. Here, we analyzed the function of Xenopus Suppressor of Hairless (XSu(H)) using morpholino antisense oligonucleotides (MO), which interfere with the translation of transcripts. In Xenopus embryos, maternal transcripts of both XSu(H)1 and XSu(H)2 were ubiquitously observed until the blastula stage and thereafter only XSu(H)1 was zygotically transcribed. Knockdown experiments with MO demonstrated that XSu(H)2 depletion caused a decrease in the expression of the Xbrachyury, MyoD and JNK1 genes. Morphological and histological examinations indicated that XSu(H)2 depletion caused abnormal gastrulation, which resulted in severe defects of the notochord and somitic mesoderm. The effect of XSu(H)2-MO was completely rescued by co-injection of XSu(H)2 mRNAs, but not by XSu(H)1 mRNAs. XESR-1, a Notch signaling target gene, inhibited Xbrachyury expression. However, expression of the XESR-1 gene was not induced by depletion of XSu(H)2. Co-injection of the dominant-negative form of XESR-1 could not rescue the suppression of Xbrachyury expression in the XSu(H)2-depleted embryo. These results suggest that XSu(H)2 is involved in mesoderm formation and the cell movement of gastrula embryos in a different manner from the XESR-1-mediated Notch signaling pathway.

XMam1, Xenopus Mastermind1, induces neural gene expression in a Notch-independent manner.

Mastermind, which is a Notch signal component, is a nuclear protein and is thought to contribute to the transactivation of target genes. Previously we showed that XMam1, Xenopus Mastermind1, was essential in the transactivation of a Notch target gene, XESR-1, and was involved in primary neurogenesis. To examine the function of XMam1 during Xenopus early development in detail, XMam1-overexpressed embryos were analyzed. Overexpression of XMam1 ectopically caused the formation of a cell mass with pigmentation on the surface of embryos and expressed nrp-1. The nrp-1-positive cell mass was produced by XMam1 without expression of the Notch target gene, XESR-1, and not by the activation form of Notch, NICD. The ectopic expression of nrp-1 was not inhibited by co-injection of XMam1 with a molecule known to inhibit Notch signaling. The nrp-1 expression was also recognized in the animal cap injected with XMam1DeltaN, which lacks the basic domain necessary for interacting with NICD and Su(H). These results show that XMam1 has the ability to induce the cell fate into the neurogenic lineage in a Notch-independent manner.

XMam1, the Xenopus homologue of mastermind, is essential to primary neurogenesis in Xenopus laevis embryos.

Notch signaling is involved in cell fate determination and is evolutionally highly conserved in vertebrates and invertebrates. Mastermind is a nuclear protein which participates in Notch signaling and is involved in direct transactivation of target genes. Here we analyzed the expression and the function of Xenopus mastermind1 (XMam1) in the process of primary neurogenesis. XMam1 is 3,425 bp and encodes 1,139 amino acids. Overall, Mastermind proteins consist of a basic domain, two acidic domains and a glutamine-rich domain, which are highly conserved among species. The ubiquitous expression of XMam1 was observed in both maternal and zygotic stages. Whole-mount in situ hybridization showed that XMam1 mRNA was present in the ectoderm by the gastrula stage and localized at the anterior neural region in the neurula stage. Thereafter, XMam1 expression was restricted to the eye and otic vesicle in the tailbud-stage embryo. XMaml overexpression caused the repression of primary neural formation. The truncated form of XMam1 (lacking the C-terminus of XMam1; XMam1deltaC) led to excess formation of primary neurons. Furthermore, XMam1deltaC strongly repressed XESR-1 transactivation. These results show that XMaml is involved in primary neurogenesis by way of Notch signaling and is an essential component for transactivation of XESR-1 in Xenopus laevis embryos.

cDNA cloning and distribution of XEXT1, the Xenopus homologue of EXT1.

Hereditary Multiple Exostosis (EXT) is an autosomal dominant disorder. Here, we have isolated XEXT1, a Xenopus homologue of EXT1, as an ovary-enriched cDNA clone. The 2,598-bp XEXT1 cDNA had a single open reading frame encoding 735 amino acids. Quantitative RT-PCR analysis showed that transcripts of XEXT1 were present maternally and consumed prior to gastrulation. Zygotic expression of XEXT1 was not detected during late embryogenesis. In adult organs, XEXT1 was expressed intensely in bone and lung. Whole-mount in situ hybridization showed that maternally transcribed XEXT1 mRNAs were stored in the animal hemisphere, and were localized in the center of the cell during cleavage stages.