An In Situ Hybridization Study of Perlecan, DMP1, and MEPE in Developing Condylar Cartilage of the Fetal Mouse Mandible and Limb Bud Cartilage

<p>The main purpose of this <em>in situ</em> hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (<em>perlecan, DMP1</em>, and <em>MEPE)</em> in developing primary (tibial) and secondary (condylar) cartilage. <em>Perlecan</em> mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, <em>perlecan</em> mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. <em>DMP1</em> mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, <em>DMP1</em> mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 <em>may</em> be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. <em>MEPE</em> mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of <em>in situ</em> hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.</p

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無線搬送式で小型・軽量タイプの心拍数モニターであるスポーツテスターPE-3000の信頼性を, 種々の運動負荷のもとで検討し, 以下の結果を得た。1)スポーツテスターの60秒モードで記憶した心拍数は, 心拍数の変化幅の大きい運動初期や運動直後に, 実際の心拍数と比較して過大評価あるいは過小評価する傾向が認められた。この傾向は, 運動強度が強くなるほど増大し, 特に1分毎の激しい間欠運動では顕著となった。2)15秒モードで得た心拍数は, 実際の心拍数変化に比較的近い値を示したが, 過大評価や過小評価の傾向はわずかに認められた。3)5秒モードによる結果は, 運動負荷の形態が異なっても, また強度の増減が激しい場合においても, 実際の心拍数と同じ値を示した。4)このような結果の差異は, 記憶時間内の平均心拍数がメモリーされるのではなく, 常にその時点での最新の心拍数が記憶されることが原因であることが明らかとなった。以上のことから, 忠実に運動時の心拍数変化を記録することが必要な場合には, より短い記憶モードを使用すべきである。しかし, 長時間の心拍応答の変化パターンが明らかになればよいという場合には, 60秒モードの使用も有効であろう。いずれにしても, 特性と限界を認識し, 目的に応じて使用するならば, スポーツテスターPE-3000は, 心拍数モニターとして非常に有益な装置である。The validity of heart rates obtained by the Sports tester PE-3000 was assessed by comparing the measuring values with simultaneous ECG recordings. One subject performed several exercise tests on a bicycle ergometer. Heart rates recorded before, during and after exercise. Under the conditions of increasing heart rate, heart rates recorded by the Sport tester PE-3000\u27s 60-seconds-mode were frequently higher than those measured at the same time by ECG. While, under the conditions of decreasing heart rate, heart rates obtained by the Sport tester were lower than those of ECG. This tendency was increase with increasing work load. However, heart rates recorded by the Sport tester\u27s 15-seconds-mode were closely related to those measured by ECG. In this condition, heart rates differed at most by only 3 beats min^. Furthermore, heart rates recorded by the Sport tester\u27s 5-seconds-mode were equal to those measured by ECG. These differences were caused mainly by the memory methods of the Sport tester PE-3000. Therefore, the shorter memory mode is suitable for measuring accurate heart rate and analyzing intensity of exercise

Soft Substrates Promote Homogeneous Self-Renewal of Embryonic Stem Cells via Downregulating Cell-Matrix Tractions

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions

BCL11B Regulates Epithelial Proliferation and Asymmetric Development of the Mouse Mandibular Incisor

Mouse incisors grow continuously throughout life with enamel deposition uniquely on the outer, or labial, side of the tooth. Asymmetric enamel deposition is due to the presence of enamel-secreting ameloblasts exclusively within the labial epithelium of the incisor. We have previously shown that mice lacking the transcription factor BCL11B/CTIP2 (BCL11B hereafter) exhibit severely disrupted ameloblast formation in the developing incisor. We now report that BCL11B is a key factor controlling epithelial proliferation and overall developmental asymmetry of the mouse incisor: BCL11B is necessary for proliferation of the labial epithelium and development of the epithelial stem cell niche, which gives rise to ameloblasts; conversely, BCL11B suppresses epithelial proliferation, and development of stem cells and ameloblasts on the inner, or lingual, side of the incisor. This bidirectional action of BCL11B in the incisor epithelia appears responsible for the asymmetry of ameloblast localization in developing incisor. Underlying these spatio-specific functions of BCL11B in incisor development is the regulation of a large gene network comprised of genes encoding several members of the FGF and TGFβ superfamilies, Sprouty proteins, and Sonic hedgehog. Our data integrate BCL11B into these pathways during incisor development and reveal the molecular mechanisms that underlie phenotypes of both Bcl11b−/− and Sprouty mutant mice

Short-term serum-free culture reveals that inhibition of Gsk3β induces the tumor-like growth of mouse embryonic stem cells.

Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3β. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3β, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3β in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3β governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs