Vitamin C transporter SVCT1 serves a physiological role as a urate importer: functional analyses and in vivo investigations.

Uric acid, the end product of purine metabolism in humans, is crucial because of its anti-oxidant activity and a causal relationship with hyperuricemia and gout. Several physiologically important urate transporters regulate this water-soluble metabolite in the human body; however, the existence of latent transporters has been suggested in the literature. We focused on the Escherichia coli urate transporter YgfU, a nucleobase-ascorbate transporter (NAT) family member, to address this issue. Only SLC23A proteins are members of the NAT family in humans. Based on the amino acid sequence similarity to YgfU, we hypothesized that SLC23A1, also known as sodium-dependent vitamin C transporter 1 (SVCT1), might be a urate transporter. First, we identified human SVCT1 and mouse Svct1 as sodium-dependent low-affinity/high-capacity urate transporters using mammalian cell-based transport assays. Next, using the CRISPR-Cas9 system followed by the crossing of mice, we generated Svct1 knockout mice lacking both urate transporter 1 and uricase. In the hyperuricemic mice model, serum urate levels were lower than controls, suggesting that Svct1 disruption could reduce serum urate. Given that Svct1 physiologically functions as a renal vitamin C re-absorber, it could also be involved in urate re-uptake from urine, though additional studies are required to obtain deeper insights into the underlying mechanisms. Our findings regarding the dual-substrate specificity of SVCT1 expand the understanding of urate handling systems and functional evolutionary changes in NAT family proteins.

Identification of GLUT12/SLC2A12 as a urate transporter that regulates the blood urate level in hyperuricemia model mice.

Recent genome-wide association studies have revealed some genetic loci associated with serum uric acid levels and susceptibility to gout/hyperuricemia which contain potential candidates of physiologically important urate transporters. One of these novel loci is located upstream of SGK1 and SLC2A12, suggesting that variations in these genes increase the risks of hyperuricemia and gout. We herein focused on SLC2A12 encoding a transporter, GLUT12, the physiological function of which remains unclear. As GLUT12 belongs to the same protein family as a well-recognized urate transporter GLUT9, we hypothesized that GLUT12 mediates membrane transport of urate. Therefore, we conducted functional assays and analyzed Glut12 knockout hyperuricemia model mice, generated using the CRISPR-Cas9 system. Our results revealed that GLUT12 acts as a physiological urate transporter and its dysfunction elevates the blood urate concentration. This study provides insights into the deeper understanding of the urate regulatory system in the body, which is also important for pathophysiology of gout/hyperuricemia.

Therapeutic efficacy of gentle endometrial curettage on antibiotic-resistant chronic endometritis in infertile women.

Purpose: To identify the efficacy of endometrial curettage on antibiotic-resistant chronic endometritis (CE) in infertile women. Methods: Of 1580 women with CE, 87 with antibiotic-resistant CE after two to five cycles of antibiotic treatment were recruited between 2019 and 2021. The women who underwent endometrial curettage without applying any force and, in the subsequent menstrual cycle, endometrial sampling for CD138 immunostaining without antibiotic use. Pregnancy outcomes after in vitro fertilization treatment were analyzed in women who did not desire endometrial curettage and in those with cured and persistent CE after endometrial curettage. Results: In 64 women who underwent endometrial curettage, the number of CD138-positive cells decreased from 28.0 ± 35.3 to 7.7 ± 14.0 (p < 0.0001), and CE in 41 women (64.1%) was cured (<5 CD138-positive cells). The pathological findings detected 3.1% of endometrial hyperplasia and 1.6% of endometrial cancer. The ongoing pregnancy rates in women aged ≤42 without endometrial curettage were significantly lower than those of women with cured and persistent CE (26.7%, 67.6%, and 57.1%, respectively, p = 0.03). Conclusions: Gentle endometrial curettage for antibiotic-resistant CE significantly decreased the number of CD138-positive cells, resulting in improved pregnancy outcomes regardless of remaining CE. Endometrial curettage is also important as a screening for endometrial malignancy.

Therapeutic effects of an oral gonadotropin-releasing hormone receptor antagonist, relugolix, on preventing premature ovulation in mild ovarian stimulation for IVF.

PURPOSE: Can relugolix, a novel oral gonadotropin-releasing hormone receptor (GnRH) antagonist, function as an alternative ovulation inhibitor to GnRH antagonist injections? METHODS: This single-center, cross-sectional retrospective study compared premature ovulation rates and clinical outcomes in IVF treatment after mild ovarian stimulation with 40 mg of relugolix (relugolix group) or 0.25-mg injections of ganirelix acetate or cetrorelix acetate (injection group) between March 2019 and January 2020. Of 247 infertile women (256 IVF cycles) aged ≤42 years, 223 women (230 cycles) were evaluated. In the relugolix and injection groups, we compared 104 and 85 cycles after GnRH antagonist use before the LH surge (LH levels <10 mIU/ml) and 22 and 19 cycles during the LH surge (LH levels ≥10 mIU/ml), respectively. RESULTS: Before the LH surge, the ovulation rates in the two groups were very low (p = 0.838), however; during the LH surge, the cycles using relugolix had a high ovulation rate of 40.9% compared with no ovulation in the injection group (p = 0.002). There were no significant differences in embryo culture findings and pregnancy outcomes between the two groups. CONCLUSIONS: Although relugolix had a high ovulation suppressive effect, when the LH surge occurred, its effect was insufficient.

Birth of a marmoset following injection of elongated spermatid from a prepubertal male.

The common marmoset is a small nonhuman primate in which the application of transgenesis and genetic knockout techniques allows the generation of gene-modified models of human diseases. However, its longer generation time than that of rodents is a major obstacle to the widespread use of gene-modified marmosets for biomedical research. In this study, we examined the feasibility of shortening the generation time by using prepubertal marmoset males as gamete donors. We collected late round stage spermatids (Steps 5-7), elongated spermatids, and testicular spermatozoa from the testis of a prepubertal 11-month-old male marmoset and injected them into in vitro-matured oocytes. After 7 days in culture, two embryos from elongated spermatid injection and two embryos from sperm injection were transferred into two separate recipient females. The recipient female that received elongated spermatid injection-derived embryos became pregnant and gave birth to one female infant. This is the first demonstration that a spermatid from a prepubertal male primate can support full-term development. Using this method, we can expect to obtain offspring of gene-modified males 6 months to a year earlier than with natural mating.

Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm.

The behavior of visceral endoderm cells was examined as the anterior visceral endoderm (AVE) formed from the distal visceral endoderm (DVE) using the mouse lines R26-H2B-EGFP and R26-PHA7-EGFP to visualize cell nuclei and adherens junction, respectively. The analysis using R26-H2B-EGFP demonstrated global cell rearrangement that was not specific to the DVE cells in the monolayer embryonic visceral endoderm sheet; each population of the endoderm cells moved collectively in a swirling movement as a whole. Most of the AVE cells at E6.5 were not E5.5 DVE cells but were E5.5 cells that were located caudally behind them, as previously reported (Hoshino et al., 2015; Takaoka et al., 2011). In the rearrangement, the posterior embryonic visceral endoderm cells did not move, as extraembryonic visceral endoderm cells did not, and they constituted a distinct population during the process of anterior-posterior axis formation. The analysis using R26-PHA7-EGFP suggested that constriction of the apical surfaces of the cells in prospective anterior portion of the DVE initiated the global cellular movement of the embryonic visceral endoderm to drive AVE formation.

Identifying a possible factor for the increased newborn size in singleton pregnancies after assisted reproductive technology using cryopreserved embryos, in comparison with fresh embryos.

PURPOSE: To determine whether the cycle regimens that are used for endometrial preparation are associated with the birthweight (BW) after assisted reproductive technology (ART) using frozen-thawed embryo transfer (FET). METHODS: The BW of singletons who were born by ART using FET was compared retrospectively, according to whether a FET was conducted in a hormone replacement therapy cycle (HRT, n = 403) or an ovulatory cycle (OVL, n = 117). The BW after timed intercourse (NAT, n = 162) also was investigated. RESULTS: There were no significant differences in the age of the mothers, percentage of primiparas, gestational periods, Body Mass Index, and sex ratio between the HRT and OVL cycles. The average BW from HRT was significantly greater than that of OVL. The BW from HRT was also greater, compared with NAT, while statistical significance was not achieved between OVL and NAT. The putative factors affecting the BW, such as ovarian stimulation protocols, endometrial thickness, and the stage and quality of embryos, could not explain the difference in the BW between the HRT and OVL cycles. CONCLUSION: An increased BW from ART using FET seems to be ascribable to conditions of the endometrium, but not cryopreservation procedures per se, which might provide a mechanistic framework for understanding heavier neonates who are born by FET.

R26-WntVis reporter mice showing graded response to Wnt signal levels.

The canonical Wnt signaling pathway plays a major role in the regulation of embryogenesis and organogenesis, where signal strength-dependent cellular responses are of particular importance. To assess Wnt signal levels in individual cells, and to circumvent the integration site-dependent bias shown in previous Wnt reporter lines, we constructed a new Wnt signal reporter mouse line R26-WntVis. Heptameric TCF/LEF1 binding sequences were combined with a viral minimal promoter to confer a graded response to the reporter depending on Wnt signal strengths. The histone H2B-EGFP fusion protein was chosen as the fluorescent reporter to facilitate single-cell resolution analyses. This WntVis reporter gene was then inserted into the ROSA26 locus in an orientation opposite to that of the endogenous gene. The R26-WntVis allele was introduced into Wnt3a(-/-) and Wnt3a(vt/-) mutant mouse embryos and compared with wild-type embryos to assess its performance. The R26-WntVis reporter was activated in known Wnt-dependent tissues and responded in a graded fashion to signal intensity. This analysis also indicated that the major Wnt activity early in embryogenesis switched from Wnt3 to Wnt3a around E7.5. The R26-WntVis mouse line will be widely useful for the study of Wnt signal-dependent processes.

Intrinsic transition of embryonic stem-cell differentiation into neural progenitors.

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.

A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes.

The CRISPR/Cas system has rapidly emerged recently as a new tool for genome engineering, and is expected to allow for controlled manipulation of specific genomic elements in a variety of species. A number of recent studies have reported the use of CRISPR/Cas for gene disruption (knockout) or targeted insertion of foreign DNA elements (knock-in). Despite the ease of simple gene knockout and small insertions or nucleotide substitutions in mouse zygotes by the CRISPR/Cas system, targeted insertion of large DNA elements remains an apparent challenge. Here the generation of knock-in mice with successful targeted insertion of large donor DNA elements ranged from 3.0 to 7.1 kb at the ROSA26 locus using the CRISPR/Cas system was achieved. Multiple independent knock-in founder mice were obtained by injection of hCas9 mRNA/sgRNA/donor vector mixtures into the cytoplasm of C57BL/6N zygotes when the injected zygotes were treated with an inhibitor of actin polymerization, cytochalasin. Successful germ line transmission of three of these knock-in alleles was also confirmed. The results suggested that treatment of zygotes with actin polymerization inhibitors following microinjection could be a viable method to facilitate targeted insertion of large DNA elements by the CRISPR/Cas system, enabling targeted knock-in readily attainable in zygotes.

Maintenance of stereocilia and apical junctional complexes by Cdc42 in cochlear hair cells.

Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre;Cdc42(flox/flox) mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.

Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter.

Fucci technology makes possible the distinction between live cells in the G(1) and S/G(2)/M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G(1) and S/G(2)/M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G(1) and S/G(2)/M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.

Case of ovarian hyperstimulation and oocyte pick-up during very early period of unnoticed pregnancy followed by ongoing normal pregnancy.

We report a case of unnoticed pregnancy that was maintained during low estrogen and progesterone circumstances, that showed menses-like bleeding, and was then discovered after ovarian hyperstimulation during the next period. The patient was 39 years old and primigravid. She underwent intrauterine insemination, followed by luteal support with human chorionic gonadotrophin and progestin; however, she experienced menstruation-like bleeding 15 days later. As low estradiol and progesterone levels were confirmed on the 2nd day of bleeding, ovarian hyperstimulation of short protocol for in vitro fertilization was commenced. Although 13 mature follicles were observed, only six oocytes were retrieved and one developed into a blastocyst. Four days after oocyte pick-up, a gestational sac was seen in utero. The fetus is currently growing uneventfully. This case suggests that pregnancy can be maintained during ovarian hyperstimulation, even if menstruation-like bleeding is shown in low-progesterone circumstances.

The synaptic targeting of mGluR1 by its carboxyl-terminal domain is crucial for cerebellar function.

The metabotropic glutamate receptor subtype 1 (mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca(2+) release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical (TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca(2+) release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.

Phosphatidic acid (PA)-preferring phospholipase A1 regulates mitochondrial dynamics.

Recent studies have suggested that phosphatidic acid (PA), a cone-shaped phospholipid that can generate negative curvature of lipid membranes, participates in mitochondrial fusion. However, precise mechanisms underling the production and consumption of PA on the mitochondrial surface are not fully understood. Phosphatidic acid-preferring phospholipase A1 (PA-PLA1)/DDHD1 is the first identified intracellular phospholipase A1 and preferentially hydrolyzes PA in vitro. Its cellular and physiological functions have not been elucidated. In this study, we show that PA-PLA1 regulates mitochondrial dynamics. PA-PLA1, when ectopically expressed in HeLa cells, induced mitochondrial fragmentation, whereas its depletion caused mitochondrial elongation. The effects of PA-PLA1 on mitochondrial morphology appear to counteract those of MitoPLD, a mitochondrion-localized phospholipase D that produces PA from cardiolipin. Consistent with high levels of expression of PA-PLA1 in testis, PA-PLA1 knock-out mice have a defect in sperm formation. In PA-PLA1-deficient sperm, the mitochondrial structure is disorganized, and an abnormal gap structure exists between the middle and principal pieces. A flagellum is bent at that position, leading to a loss of motility. Our results suggest a possible mechanism of PA regulation of the mitochondrial membrane and demonstrate an in vivo function of PA-PLA1 in the organization of mitochondria during spermiogenesis.

Insulin resistance in nonobese Japanese women with polycystic ovary syndrome is associated with poorer glucose tolerance, delayed insulin secretion, and enhanced insulin response.

Purpose: To determine the prevalence of insulin resistance (IR) and impaired glucose tolerance (IGT) in PCOS patients, the optimal screening method, and to compare our findings between nonobese and obese Japanese women with PCOS. Methods: Ninety-eight PCOS patients were included in this research from 2006 to 2013. Glucose tolerance test (OGTT) was performed. Serum glucose and insulin concentration were assayed before and 30, 60, and 120 min after taking 75 g of glucose. Results: All examined metabolic parameters were significantly favorable in the nonobese subjects, below 25 kg/m2. HOMA-IR, fasting insulin, glucose120, and insulin120 showed strong correlations with BMI. A total of 1.4 % of nonobese women had IR based on fasting insulin or HOMA-IR. However, 15.5 % (11/71) of nonobese women had IR as determined by a continuous increase of serum insulin level in OGTT. In comparison, the prevalence of IR among the obese women ranged from 41 to 59 %. AUCglucose, glucose60, glucose120, and insulin120 in nonobese women with a continuous insulin increase were higher than those without such a continuous increase. Conclusions: All examined metabolic parameters were significantly correlated with BMI. As the presence of a continuous increase of insulin level reflects to some degree poorer glucose tolerance, delayed insulin secretion, and enhanced insulin response compared with non-continuous insulin increase, OGTT might not been excluded to determine IR and IGT for nonobese women with PCOS.

The glutamate receptor GluN2 subunit regulates synaptic trafficking of AMPA receptors in the neonatal mouse brain.

The N-methyl-D-aspartate receptor (NMDAR) plays various physiological and pathological roles in neural development, synaptic plasticity and neuronal cell death. It is composed of two GluN1 and two GluN2 subunits and, in the neonatal hippocampus, most synaptic NMDARs are GluN2B-containing receptors, which are gradually replaced with GluN2A-containing receptors during development. Here, we examined whether GluN2A could be substituted for GluN2B in neural development and functions by analysing knock-in (KI) mice in which GluN2B is replaced with GluN2A. The KI mutation was neonatally lethal, although GluN2A-containing receptors were transported to the postsynaptic membrane even without GluN2B and functional at synapses of acute hippocampal slices of postnatal day 0, indicating that GluN2A-containing NMDARs could not be substituted for GluN2B-containing NMDARs. Importantly, the synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluA1 was increased, and the transmembrane AMPAR regulatory protein, which is involved in AMPAR synaptic trafficking, was increased in KI mice. Although the regulation of AMPARs by GluN2B has been reported in cultured neurons, we showed here that AMPAR-mediated synaptic responses were increased in acute KI slices, suggesting differential roles of GluN2A and GluN2B in AMPAR expression and trafficking in vivo. Taken together, our results suggest that GluN2B is essential for the survival of animals, and that the GluN2B-GluN2A switching plays a critical role in synaptic integration of AMPARs through regulation of GluA1 in the whole animal.

Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis.

The transition from mitosis to meiosis is unique to germ cells. In murine embryonic ovaries and juvenile testes, retinoic acid (RA) induces meiosis via the stimulated by retinoic acid gene 8 (Stra8), but its molecular pathway requires elucidation. We present genetic evidence in vivo and in vitro that neuregulins (NRGs) are essential for the proliferation of spermatogonia and the initiation of meiosis. Tamoxifen (TAM) was injected into 14-day post-partum (dpp) Sertoli cell-specific conditional Nrg1(Ser-/-) mutant mice. TAM induced testis degeneration, suppressed BrdU incorporation into spermatogonia and pre-leptotene primary spermatocytes, and decreased and increased the number of STRA8-positive and TUNEL-positive cells, respectively. In testicular organ cultures from 5-6 dpp wild-type mice and cultures of their re-aggregated spermatogonia and Sertoli cells, FSH, RA [all-trans-retinoic acid (ATRA), AM580, 9-cis-RA] and NRG1 promoted spermatogonial proliferation and meiotic initiation. However, TAM treatment of testicular organ cultures from the Nrg1(Ser-/-) mutants suppressed spermatogonial proliferation and meiotic initiation that was promoted by FSH or AM580. In re-aggregated cultures of purified spermatogonia, NRG1, NRG3, ATRA and 9-cis-RA promoted their proliferation and meiotic initiation, but neither AM580 nor FSH did. In addition, FSH, RAs and NRG1 promoted Nrg1 and Nrg3 mRNA expression in Sertoli cells. These results indicate that in juvenile testes RA and FSH induced meiosis indirectly through Sertoli cells when NRG1 and NRG3 were upregulated, as NRG1 amplified itself and NRG3. The amplified NRG1 and NRG3 directly induced meiosis in spermatogonia. In addition, ATRA and 9-cis-RA activated spermatogonia directly and promoted their proliferation and eventually meiotic initiation.

Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods.

The Six1 homeobox gene plays critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in human SIX1 cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing loss and branchial defects. The present study was designed to identify enhancers responsible for the dynamic expression pattern of Six1 during mouse embryogenesis. The results showed distinct enhancer activities of seven conserved non-coding sequences (CNSs) retained in tetrapod Six1 loci. The activities were detected in all cranial placodes (excluding the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-expression domains during development were covered by the sum of activities of these enhancers, together with the previously identified enhancer for the pre-placodal region and foregut endoderm. Thus, the eight CNSs identified in a series of our study represent major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. The results also confirmed that chick electroporation is a robust means to decipher regulatory information stored in vertebrate genomes. Mutational analysis of the most conserved placode-specific enhancer, Six1-21, indicated that the enhancer integrates a variety of inputs from Sox, Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. Positive autoregulation of Six1 is achieved through the regulation of Six protein-binding sites. The identified Six1 enhancers provide valuable tools to understand the mechanism of Six1 regulation and to manipulate gene expression in the developing embryo, particularly in the sensory organs.

Phf14, a novel regulator of mesenchyme growth via platelet-derived growth factor (PDGF) receptor-α.

The regulation of mesenchymal cell growth by signaling molecules plays an important role in maintaining tissue functions. Aberrant mesenchymal cell proliferation caused by disruption of this regulatory process leads to pathogenetic events such as fibrosis. In the current study we have identified a novel nuclear factor, Phf14, which controls the proliferation of mesenchymal cells by regulating PDGFRα expression. Phf14-null mice died just after birth due to respiratory failure. Histological analyses of the lungs of these mice showed interstitial hyperplasia with an increased number of PDGFRα(+) mesenchymal cells. PDGFRα expression was elevated in Phf14-null mesenchymal fibroblasts, resulting in increased proliferation. We demonstrated that Phf14 acts as a transcription factor that directly represses PDGFRα expression. Based on these results, we used an antibody against PDGFRα to successfully treat mouse lung fibrosis. This study shows that Phf14 acts as a negative regulator of PDGFRα expression in mesenchymal cells undergoing normal and abnormal proliferation, and is a potential target for new treatments of lung fibrosis.