Fine mapping of the circling (cir) gene on the distal portion of mouse chromosome 9

Circling mice manifest profound deafness, head-tossing, and bi-directional circling behavior, which they inherit in autosomal recessive manner. Histologic examination of the inner ear reveals abnormalities of the region around the organ of Corti, spiral ganglion neurons, and outer hair cells. A genetic linkage map was constructed for an intraspecific backcross between cir and C57BL/6J mice. The cir gene was mapped to a region between D9Mit116/D9Mit15 and D9Mit38 on mouse chromosome (Chr) 9. Estimated distances between cir and D9Mit116, and between cir and D9Mit38 were 0.70 +/- 0.40 and 0.23 +/- 0.23 cM, respectively. Order of the markers was defined as follows: centromere - D9Mit182 - D9Mit51/D9Mit79/D9Mit310 - D9Mit212/D184 - D9Mit116/D9Mit15 - cir - D9Mit38 - D9Mit20 - D9Mit243 - D9Mit16 - D9Mit55/D9Mit125 - D9Mit281. On the basis of genetic mapping, we constructed a yeast artificial chromosome (YAC) contig across the cir region. The cir gene is located between the lactotransferrin (ltf) and microtubule-associated protein (map4) genes. The distal portion of mouse Chr 9 encompassing the cir region is homologous with human chromosome 3p21, which contains the Deafness, form B: Autosomal Recessive Deafness (DFNB6) locus. Therefore, the circling mouse is a potential animal model for DFNB6 deafness in humans.     

Tetrachloroethylene, trichloroethylene, and chlorinated phenols induce toluene-o-xylene monooxygenase activity in Pseudomonas stutzeri OX1

Pseudomonas stutzeri OX1 naphthalene-oxidation activity is induced 3.0-fold by tetrachloroethylene (PCE) and 3.1-fold by trichloroethylene (TCE) at 100 microM. With the mutant P. stutzeri M1, which does not express toluene-o-xylene monooxygenase (ToMO, product of the tou operon), no naphthalene-oxidation activity induction by PCE and TCE was found; hence, PCE and TCE induce ToMO of P. stutzeri OX1. The chlorinated phenols 2-, 3-, and 4-chlorophenol induced ToMO expression 0.58-, 0.23- and 0.37-fold, respectively, compared to the direct inducer of the pathway, o-cresol. Using P. putida PaW340 (pPP4062, pFP3028), which has the tou promoter fused to the reporter catechol-2,3-dioxygenase, and the regulator gene touR, it was determined that the tou promoter was induced directly 5.7-, 7.1-, and 5.1-fold for 2-, 3-, and 4-chlorophenol, respectively (compared to an 8.8-fold induction with o-cresol). In addition, it was found that TCE and PCE do not directly induce the tou pathway and that components other than the tou structural and regulatory genes are necessary for induction. Gas chromatography results also showed that 100 microM TCE induced its own degradation (8-9%) in 16 h in P. stutzeri OX1, and all of the stoichiometric chloride from the degraded TCE was detected in solution.     

Epidermal growth factor receptor regulates osteoclast differentiation and survival through cross-talking with RANK signaling

The epidermal growth factor receptor (EGFR) functions in various cellular physiological processes such as proliferation, differentiation, and motility. Although recent studies have reported that EGFR signaling is involved in osteoclast recruitment and formation, the molecular mechanism of EGFR signaling for the regulation of osteoclastogenesis remains unclear. We investigated the role of the EGFR in osteoclast differentiation and survival and show that the expression of the EGFR was highly up-regulated by receptor activator of nuclear factor-kappaB ligand (RANKL) during osteoclast differentiation. EGFR-specific tyrosine kinase inhibitors and EGFR knockdown blocked RANKL-dependent osteoclast formation, suggesting that EGFR signaling plays an important role in osteoclastogenesis. EGFR inhibition impaired the RANKL-mediated activation of osteoclastogenic signaling pathways, including c-Jun N-terminal kinase (JNK), NF-kappaB, and Akt/protein kinase B (PKB). In addition, EGFR inhibition in differentiated osteoclasts caused apoptosis through caspase activation. Inhibition of the phosphoinositide-3 kinase (PI3K)-Akt/PKB pathway and subsequent activation of BAD and caspases-9 and -3 may be responsible for the EGFR inhibition-induced apoptosis. The EGFR physically associated with receptor activator of nuclear factor-kappaB (RANK) and Grb2-associated binder 2 (Gab2). Moreover, RANKL transactivated EGFR. These data indicate that EGFR regulates RANKL-activated signaling pathways by cross-talking with RANK, suggesting that the EGFR may play a crucial role as a RANK downstream signal and/or a novel type of RANK co-receptor in osteoclast differentiation and survival.     

A TGF‐β‐induced gene, βig‐h3, is crucial for the apoptotic disappearance of the medial edge epithelium in palate fusion

TGF‐β3, TβR‐I, and TGF‐β‐activated Smad2 has been suggested to be a series of signaling molecules for secondary palate fusion. In this article, we show that a gene induced by TGF‐β, βig‐h3, is coincidentally expressed with TGF‐β3 in medial edge epithelial (MEE) cells undergoing apoptosis during normal palatal fusion. βig‐h3 was also highly expressed in the areas of post‐weaning mammary gland cells and developing phalangeal joints in which TGF‐β3 or BMP‐4‐induced apoptosis occurs, respectively. Blocking of βig‐h3 expression in E12.5 embryos with antisense oligodeoxynucleotides (ODN) resulted in cleft of the secondary palate in 84% of the treated mice that were born. Moreover, the antisense ODN treatment resulted in a failure of apoptosis in the MEE between palatal shelves in physical contact in organ culture. We conclude that βig‐h3 expression in the MEE is stimulated by TGF‐β3, causes cell death, and consequently results in complete fusion of the apposed palatal shelves. J. Cell. Biochem. 107: 818–825, 2009. © 2009 Wiley‐Liss, Inc.     

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.     

TGF-beta2 stimulates cranial suture closure through activation of the Erk-MAPK pathway

Cranial sutures are important growth sites of the skull. During suture closure, the dura mater is one of the most important sources of various positive and negative regulatory signals. Previous results indicate that TGF-beta2 from dura mater strongly accelerates suture closure, however, its exact regulatory mechanism is still unclear. In this study, we confirmed that removal of dura mater in calvarial organ culture strongly accelerates sagittal suture closure and that this effect is further enhanced by TGF-beta2 treatment. TGF-beta2 stimulated cell proliferation in the MC3T3-E1 cell line. Similarly, it stimulated the proliferation of cells in the sutural space in calvarial organ culture. Furthermore, TGF-beta2-mediated enhanced cell proliferation and suture closure were almost completely inhibited by an Erk-MAPK blocker, PD98059. These results indicate that TGF-beta2-induced activation of Erk-MAPK is an important signaling component that stimulates cell proliferation to enrich osteoprogenitor cells, thereby promoting their differentiation into osteoblasts to achieve a rapid calvarial bone expansion.     

Spatiotemporal Expression of tmie in the Inner Ear of Rats during Postnatal Development

The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6. Transmembrane inner ear (tmie) is the causative gene and its mutation through deletion of a 40-kilobase genomic region including tmie leads to deafness. The function of Tmie is unknown. To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie in the rat cochlea by using a Tmie-specific antibody. Results showed that tmie expression was prominent in early postnatal rat cochleas in the stereocilia bundles of hair cells. The Tmie signal spread from the stereocilia to the hair cell body region and on to organ of Corti cells. No Tmie signal was observed in cell nuclei; Tmie was localized to the cytoplasm. Because Tmie is predicted to have 1 or 2 transmembrane domains, we postulate that it is localized to membrane-based organelles or the plasma membrane. Our results imply that Tmie exists in the cytoplasm and may have a key role in the maturation and structure of stereocilia bundles in developing hair cells. After hair cell maturation, Tmie is thought to be involved in the maintenance of organ of Corti cells.Circling is often observed in mouse and rat deafness mutants and is commonly suggested to be a consequence of inner ear defects that impair vestibular systems.^3,12,14 The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6; these mice have abnormal circling behavior, suggesting a balance disorder, and profound deafness.^6,7 The most notable pathologic phenotypes of circling mice are the almost completely degenerated cochlea and remarkably reduced cellularity in spiral ganglion neurons. The causative gene for circling is transmembrane inner ear (tmie), with a 40-kilobase genomic deletion including tmie.¹ tmie is also the causative gene of the spinner (sr/sr) mouse, which has phenotypes similar to circling mice, although the mutation patterns are different.⁸ Spinner mice also show circling behavior, hearing loss, imbalance, and swimming inability. In addition, spinner mice have 2 mutations in the tmie gene: the 40-kb genomic deletion including tmie and a point mutation that leads to a truncated protein.⁸In humans, 7 different homozygous recessive mutations in TMIE currently are known to exist in affected members of consanguineous families segregating severe-to-profound prelingual deafness, consistent with linkage to DFNB6.^9,10 Although the functions of murine Tmie and human TMIE are unknown, this protein appears to be important for normal hearing and vestibular function.In a previous study, we produced transgenic mice overexpressing tmie that resulted in phenotypic rescue of circling.¹¹ Normal expression of transgenic tmie induced phenotypic rescue in circling homozygous mutants, although some mice did not show amelioration of abnormal behavior, hearing ability, or tissue morphology in the inner ear. Therefore the Tmie protein is required for normal inner ear function in mouse.¹¹To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie. Knowing when, where, and to what extent this protein is produced in the developing inner ear will provide important clues to protein function. In adult mouse and rat, tmie is expressed in various tissues.^2,13 Whether Tmie plays an important role in those tissues is uncertain, because circling mice that lack the entire tmie gene have no noteworthy problems in any tissues except those of the inner ear systems.⁶In this study, we were interested in the postnatal stages before and after the onset of hearing (around postnatal day [P] 12) in rats; therefore, the postnatal period P0 to19 was studied. Although all the cells that form the mature cochlea are present at birth, important conformational changes occur during this period, including the formation of the tunnel of Corti and the establishment or retraction of neuronal connections. The expression pattern of tmie in the developing inner ear during early postnatal development has not been investigated previously. Here we document our use of a Tmie-specific antibody to elucidate the spatial and temporal expression of tmie in the rat inner ear during postnatal development.