Transgenic expression of Map3k4 rescues T-associated sex reversal (Tas) in mice.

Disorders of sex development in the human population range in severity from mild genital defects to gonadal sex reversal. XY female development has been associated with heterozygous mutations in several genes, including SOX9, WT1 and MAP3K1. In contrast, XY sex reversal in mice usually requires complete absence of testis-determining gene products. One exception to this involves T-associated sex reversal (Tas), a phenomenon characterized by the formation of ovotestes or ovaries in XY mice hemizygous for the hairpin-tail (T(hp)) or T-Orleans (T(Orl)) deletions on proximal mouse chromosome 17. We recently reported that mice heterozygous for a null allele of Map3k4, which resides in the T(hp) deletion, exhibit XY ovotestis development and occasional gonadal sex reversal on the sensitized C57BL/6J-Y(AKR) (B6-Y(AKR)) genetic background, reminiscent of the Tas phenotype. However, these experiments did not exclude the possibility that loss of other loci in the T(hp) deletion, or other effects of the deletion itself, might contribute to Tas. Here, we show that disruption to Sry expression underlies XY gonadal defects in B6-Y(AKR) embryos harbouring the T(hp) deletion and that a functional Map3k4 bacterial artificial chromosome rescues these abnormalities by re-establishing a normal Sry expression profile. These data demonstrate that Map3k4 haploinsufficiency is the cause of T-associated sex reversal and that levels of this signalling molecule are a major determinant of the expression profile of Sry.

Development of a general-purpose method for cell purification using Cre/loxP-mediated recombination.

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general-purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock-in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP-mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1-Cre-transgenic (tg) embryos almost equally as efficiently as from Nr5a1-hCD271-tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh-Cre-tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types.

JMJD1C, a JmjC domain-containing protein, is required for long-term maintenance of male germ cells in mice.

JmjC domain-containing proteins are a class of enzymes responsible for histone demethylation. Previous studies revealed that the JmjC domain-containing protein KDM3A possesses intrinsic demethylase activity toward lysine 9 of histone H3 and plays essential roles in spermiogenesis. In contrast, the biological roles of JMJD1C, a KDM3A homolog in mice, are largely unknown. Here we present the crucial role of JMJD1C in male gametogenesis. Jmjd1c-deficient males became infertile due to the progressive reduction of germ cells after 3 mo of age. Importantly, Jmjd1c-deficient testes frequently contained abnormal tubules lacking developmentally immature germ cells. JMJD1C is most abundantly expressed in undifferentiated spermatogonia in mouse testis. The numbers of ZBTB16-positive spermatogonia and apoptotic germ cells in Jmjd1c-deficient testes decreased and increased in an age-dependent manner, respectively. Our studies demonstrated that JMJD1C contributes to the long-term maintenance of the male germ line.

Biochemical characterization and gene expression of two endo-arabinanases from Penicillium chrysogenum 31B.

We previously described five arabinanolytic enzymes secreted by Penicillium chrysogenum 31B into the culture medium. Here, we describe a sixth such enzyme, termed AbnS1. Analysis of the reaction products of debranched arabinan revealed that AbnS1 cleaved the substrate in an endo manner. The optimum temperature of AbnS1 was 60°C, which was much higher than that of a cold-adapted endo-arabinanase (Abnc) produced by this strain. The abns1 cDNA gene encoding AbnS1 was isolated by in vitro cloning. The deduced amino acid sequence of AbnS1 had 70% identity with that of Abnc. Pfam analysis revealed a Glyco_hydro_43 domain at positions 28 to 318 of AbnS1. Semi-quantitative reverse transcription-polymerase chain reaction analysis indicated that the abns1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was only slightly induced with arabinose and arabinan. In contrast, expression of the abnc gene encoding Abnc was strongly induced by arabinose, arabinitol, and arabinan. Using debranched arabinan as substrate, recombinant AbnS1 (rAbnS1) accumulated arabinobiose and arabinotriose as the major products. Recombinant Abnc (rAbnc) released mainly arabinotriose and lesser amounts of arabinose and arabinobiose than did rAbnS1. Branched arabinan was completely degraded to arabinose by the action of rAbnS1 or rAbnc in combination with α-L: -arabinofuranosidase.

Direct conversion of fibroblasts into urothelial cells that may be recruited to regenerating mucosa of injured urinary bladder.

Urothelial cells play essential roles in protection of urine exudation and bacterial invasion at the urothelial mucosa, so that defect or damage of urothelial cells associated with urinary tract diseases may cause serious problems. If a sufficient number of functional urothelial cells are prepared in culture and transplanted into the damaged urothelial lesions, such technology may provide beneficial effects to patients with diseases of the urinary tract. Here we found that human adult dermal fibroblasts were converted into urothelial cells by transducing genes for four transcription factors, FOXA1, TP63, MYCL and KLF4 (FTLK). The directly converted urothelial cells (dUCs) formed cobblestone-like colonies and expressed urothelium-specific markers. dUCs were successfully expanded and enriched after serial passages using a specific medium that we optimized for the cells. The passaged dUCs showed similar genome-wide gene expression profiles to normal urothelial cells and had a barrier function. The FTLK-transduced fibroblasts were also converted into urothelial cells in vivo and recruited to the regenerating urothelial tissue after they were transplanted into the bladder of mice with interstitial cystitis. Our technology may provide a promising solution for a number of patients with urinary tract disorders.

Combined Loss of JMJD1A and JMJD1B Reveals Critical Roles for H3K9 Demethylation in the Maintenance of Embryonic Stem Cells and Early Embryogenesis.

Histone H3 lysine 9 (H3K9) methylation is unevenly distributed in mammalian chromosomes. However, the molecular mechanism controlling the uneven distribution and its biological significance remain to be elucidated. Here, we show that JMJD1A and JMJD1B preferentially target H3K9 demethylation of gene-dense regions of chromosomes, thereby establishing an H3K9 hypomethylation state in euchromatin. JMJD1A/JMJD1B-deficient embryos died soon after implantation accompanying epiblast cell death. Furthermore, combined loss of JMJD1A and JMJD1B caused perturbed expression of metabolic genes and rapid cell death in embryonic stem cells (ESCs). These results indicate that JMJD1A/JMJD1B-meditated H3K9 demethylation has critical roles for early embryogenesis and ESC maintenance. Finally, genetic rescue experiments clarified that H3K9 overmethylation by G9A was the cause of the cell death and perturbed gene expression of JMJD1A/JMJD1B-depleted ESCs. We summarized that JMJD1A and JMJD1B, in combination, ensure early embryogenesis and ESC viability by establishing the correct H3K9 methylated epigenome.

Role of Immediate-Early Genes in Synaptic Plasticity and Neuronal Ensembles Underlying the Memory Trace.

In the brain, neuronal gene expression is dynamically changed in response to neuronal activity. In particular, the expression of immediate-early genes (IEGs) such as egr-1, c-fos, and Arc is rapidly and selectively upregulated in subsets of neurons in specific brain regions associated with learning and memory formation. IEG expression has therefore been widely used as a molecular marker for neuronal populations that undergo plastic changes underlying formation of long-term memory. In recent years, optogenetic and pharmacogenetic studies of neurons expressing c-fos or Arc have revealed that, during learning, IEG-positive neurons encode and store information that is required for memory recall, suggesting that they may be involved in formation of the memory trace. However, despite accumulating evidence for the role of IEGs in synaptic plasticity, the molecular and cellular mechanisms associated with this process remain unclear. In this review, we first summarize recent literature concerning the role of IEG-expressing neuronal ensembles in organizing the memory trace. We then focus on the physiological significance of IEGs, especially Arc, in synaptic plasticity, and describe our hypotheses about the importance of Arc expression in various types of input-specific circuit reorganization. Finally, we offer perspectives on Arc function that would unveil the role of IEG-expressing neurons in the formation of memory traces in the hippocampus and other brain areas.

Epigenetic regulation of mouse sex determination by the histone demethylase Jmjd1a.

Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.