Video_2_Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro.MP4

Palatal fusion is a critical step during palatogenesis. In this fusing interface, the epithelial sheets need to be removed in order to achieve mesenchymal continuity. Epithelial cellular migration is one of the possible mechanisms, and live imaging of the labeled epithelium could provide direct evidence for it. However, the removal of medial edge epithelium (MEE) between the bilateral processes takes place in the middle of the dorso-ventral axis of the palatal shelf, and thus it is challenging to capture the cellular behavior directly. Here, we evaluate cellular behavior of MEE cells using a live imaging technique with a mouse model which expresses GFP under the promoter of Keratin14 (K14-GFP) and unpaired palatal shelf culture. Using this approach, we successfully obtained live images of epithelial behavior and detected epithelial cell migration on the surface of the secondary palatal shelf without touching of the opposing shelf. Additionally, the pattern of epithelial elimination resulted in oval-shaped exposed mesenchyme, which recapitulated the situation during secondary palate fusion in vivo. Detailed image processing revealed that most of the MEE migrated in an outward direction at the boundary regions as the oval shape of the exposed mesenchyme expanded. The migration was preceded by the bulging of MEE, and disappearance of GFP signals was not evident in bulging or migrating MEE at the boundary regions. Furthermore, the MEE migration and the subsequent mesenchymal exposure were disturbed by application of ROCK inhibitor. Together, these findings indicated that epithelial cell migration contributed importantly to the MEE removal and the subsequent exposure of the underlying mesenchyme. Furthermore, they indicated that the migration of epithelial cells was regulated in a time- and space-specific manner, since unpaired palatal shelf culture exhibited these cellular behaviors even in the absence of the opposing shelf. Altogether, present data indicated that this new experimental system combining live imaging with GFP-labeled epithelium mice and unpaired palatal shelf culture enabled direct visualization of cellular migration of MEE in vitro and could be a powerful tool to investigate its cellular and molecular mechanisms.</p

Image_1_Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro.TIF

Palatal fusion is a critical step during palatogenesis. In this fusing interface, the epithelial sheets need to be removed in order to achieve mesenchymal continuity. Epithelial cellular migration is one of the possible mechanisms, and live imaging of the labeled epithelium could provide direct evidence for it. However, the removal of medial edge epithelium (MEE) between the bilateral processes takes place in the middle of the dorso-ventral axis of the palatal shelf, and thus it is challenging to capture the cellular behavior directly. Here, we evaluate cellular behavior of MEE cells using a live imaging technique with a mouse model which expresses GFP under the promoter of Keratin14 (K14-GFP) and unpaired palatal shelf culture. Using this approach, we successfully obtained live images of epithelial behavior and detected epithelial cell migration on the surface of the secondary palatal shelf without touching of the opposing shelf. Additionally, the pattern of epithelial elimination resulted in oval-shaped exposed mesenchyme, which recapitulated the situation during secondary palate fusion in vivo. Detailed image processing revealed that most of the MEE migrated in an outward direction at the boundary regions as the oval shape of the exposed mesenchyme expanded. The migration was preceded by the bulging of MEE, and disappearance of GFP signals was not evident in bulging or migrating MEE at the boundary regions. Furthermore, the MEE migration and the subsequent mesenchymal exposure were disturbed by application of ROCK inhibitor. Together, these findings indicated that epithelial cell migration contributed importantly to the MEE removal and the subsequent exposure of the underlying mesenchyme. Furthermore, they indicated that the migration of epithelial cells was regulated in a time- and space-specific manner, since unpaired palatal shelf culture exhibited these cellular behaviors even in the absence of the opposing shelf. Altogether, present data indicated that this new experimental system combining live imaging with GFP-labeled epithelium mice and unpaired palatal shelf culture enabled direct visualization of cellular migration of MEE in vitro and could be a powerful tool to investigate its cellular and molecular mechanisms.</p

Video_1_Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro.MP4

Palatal fusion is a critical step during palatogenesis. In this fusing interface, the epithelial sheets need to be removed in order to achieve mesenchymal continuity. Epithelial cellular migration is one of the possible mechanisms, and live imaging of the labeled epithelium could provide direct evidence for it. However, the removal of medial edge epithelium (MEE) between the bilateral processes takes place in the middle of the dorso-ventral axis of the palatal shelf, and thus it is challenging to capture the cellular behavior directly. Here, we evaluate cellular behavior of MEE cells using a live imaging technique with a mouse model which expresses GFP under the promoter of Keratin14 (K14-GFP) and unpaired palatal shelf culture. Using this approach, we successfully obtained live images of epithelial behavior and detected epithelial cell migration on the surface of the secondary palatal shelf without touching of the opposing shelf. Additionally, the pattern of epithelial elimination resulted in oval-shaped exposed mesenchyme, which recapitulated the situation during secondary palate fusion in vivo. Detailed image processing revealed that most of the MEE migrated in an outward direction at the boundary regions as the oval shape of the exposed mesenchyme expanded. The migration was preceded by the bulging of MEE, and disappearance of GFP signals was not evident in bulging or migrating MEE at the boundary regions. Furthermore, the MEE migration and the subsequent mesenchymal exposure were disturbed by application of ROCK inhibitor. Together, these findings indicated that epithelial cell migration contributed importantly to the MEE removal and the subsequent exposure of the underlying mesenchyme. Furthermore, they indicated that the migration of epithelial cells was regulated in a time- and space-specific manner, since unpaired palatal shelf culture exhibited these cellular behaviors even in the absence of the opposing shelf. Altogether, present data indicated that this new experimental system combining live imaging with GFP-labeled epithelium mice and unpaired palatal shelf culture enabled direct visualization of cellular migration of MEE in vitro and could be a powerful tool to investigate its cellular and molecular mechanisms.</p

Video_3_Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro.MP4

Palatal fusion is a critical step during palatogenesis. In this fusing interface, the epithelial sheets need to be removed in order to achieve mesenchymal continuity. Epithelial cellular migration is one of the possible mechanisms, and live imaging of the labeled epithelium could provide direct evidence for it. However, the removal of medial edge epithelium (MEE) between the bilateral processes takes place in the middle of the dorso-ventral axis of the palatal shelf, and thus it is challenging to capture the cellular behavior directly. Here, we evaluate cellular behavior of MEE cells using a live imaging technique with a mouse model which expresses GFP under the promoter of Keratin14 (K14-GFP) and unpaired palatal shelf culture. Using this approach, we successfully obtained live images of epithelial behavior and detected epithelial cell migration on the surface of the secondary palatal shelf without touching of the opposing shelf. Additionally, the pattern of epithelial elimination resulted in oval-shaped exposed mesenchyme, which recapitulated the situation during secondary palate fusion in vivo. Detailed image processing revealed that most of the MEE migrated in an outward direction at the boundary regions as the oval shape of the exposed mesenchyme expanded. The migration was preceded by the bulging of MEE, and disappearance of GFP signals was not evident in bulging or migrating MEE at the boundary regions. Furthermore, the MEE migration and the subsequent mesenchymal exposure were disturbed by application of ROCK inhibitor. Together, these findings indicated that epithelial cell migration contributed importantly to the MEE removal and the subsequent exposure of the underlying mesenchyme. Furthermore, they indicated that the migration of epithelial cells was regulated in a time- and space-specific manner, since unpaired palatal shelf culture exhibited these cellular behaviors even in the absence of the opposing shelf. Altogether, present data indicated that this new experimental system combining live imaging with GFP-labeled epithelium mice and unpaired palatal shelf culture enabled direct visualization of cellular migration of MEE in vitro and could be a powerful tool to investigate its cellular and molecular mechanisms.</p

DS_10.1177_0363546518778789 – Supplemental material for Modulating Glucose Metabolism and Lactate Synthesis in Injured Mouse Tendons: Treatment With Dichloroacetate, a Lactate Synthesis Inhibitor, Improves Tendon Healing

<p>Supplemental material, DS_10.1177_0363546518778789 for Modulating Glucose Metabolism and Lactate Synthesis in Injured Mouse Tendons: Treatment With Dichloroacetate, a Lactate Synthesis Inhibitor, Improves Tendon Healing by Kairui Zhang, Michael W. Hast, Soutarou Izumi, Yu Usami, Snehal Shetye, Ngozi Akabudike, Nancy J. Philp, Masahiro Iwamoto, Itzhak Nissim, Louis J. Soslowsky and Motomi Enomoto-Iwamoto in The American Journal of Sports Medicine</p

Supplement_information_rev_Wilson – Supplemental material for Analysis of Association between Morphometric Parameters of Growth Plate and Bone Growth of Tibia in Mice and Humans

Supplemental material, Supplement_information_rev_Wilson for Analysis of Association between Morphometric Parameters of Growth Plate and Bone Growth of Tibia in Mice and Humans by Kimberly Wilson, Yu Usami, Danielle Hogarth, Amanda L. Scheiber, Hongying Tian, Takeshi Oichi, Yulong Wei, Ling Qin, Satoru Otsuru, Satoru Toyosawa, Masahiro Iwamoto, Joshua M. Abzug and Motomi Enomoto-Iwamoto in CARTILAGE</p

Synovial changes in control and α5 CKO mice after OA surgery.

<p>The knee joints were harvested from control (A, C, E and G) and α5 CKO (B, D, F and H) 4 weeks after OA surgery. The sagittal sections were subjected to hematoxylin-eosin staining (HE, A-D), CD31 immunostaining (E and F) and Safranin O staining (G and H). C and D are magnified images of the box of A and B, respectively. Double headed arrows indicate thickening of synovium (C and D). Arrows indicate CD31-positive vessels (E). Bar, 250 μm for A, B, G and H; 125 μm for C-F.</p

Distribution of α5 integrin in the OA and healthy knee joints.

<p>The knee joints were harvested from the surgery site (B, D, F and H) and the contralateral site (A, C, E and G) 4 weeks after OA surgery. A, B, E and F, Superimposed images of α5 integrin staining (red) with DAPI nuclear staining (blue). C, D, G and H, Superimposed images of α5 integrin staining (red) with the phase contrast images. Bar, 40 μm.</p

Apoptotic cells in control and α5 CKO articular cartilage after OA surgery.

<p>The knee joints were harvested from control (A and C) and α5 CKO (B and D) 4 weeks after OA surgery. The sagittal sections were subjected to TUNEL staining. The fluorescence images (green) were superimposed with the corresponding phase images. Bar, 40 μm. E, The ratio of the TUNEL-positive cells to total cells was calculated. The graphs represent average and SD (n = 6/group).</p

Biomechanical properties in the articular cartilage in control and α5 CKO mice.

<p>The knee joints were harvested from control and α5 CKO mice 4 weeks after OA surgery (OA surgery) and without surgery (No surgery). The femoral condyle was subjected to AFM-based nanoindentation assessment. The graph represents average and SE, n = 5/group. <i>Eind</i>, Effective indentation module calculated from Hertz model.</p