Additional file 2: of Ectopic Fgf signaling induces the intercalary response in developing chicken limb buds

Figure S2. Joint-like formation between the host stylopod and the induced zeugopod by Fgf2 + Fgf8 application. (A) Gdf5 expression. (B) Type II collagen expression. The scale bar is 2 mm. (JPEG 2568 kb

Additional file 1: of Ectopic Fgf signaling induces the intercalary response in developing chicken limb buds

Figure S1. Skeletal pattern of the intercalary regenerated chick wing by Fgf2 + Fgf8 bead grafting. Skeletal pattern was visualized by Alcian blue staining. The scale bar is 2 mm. (JPEG 1535 kb

<i>Type I</i> and <i>type II collagen</i> expression patterns of cartilage.

<p>* Only in early stage.</p><p><i>Type I</i> and <i>type II collagen</i> expression patterns of cartilage.</p

<i>Type I</i> and <i>type II collagen</i> expression patterns in the <i>Xenopus</i> limb bud.

<p>(A-C) The st. 52 <i>Xenopus</i> limb bud. (A) HE and Alcian blue staining. (B) <i>Type I collagen</i> expression. (C) <i>Type II collagen</i> expression. (D-F) The distal part of the st. 54 <i>Xenopus</i> limb bud. (D) HE and Alcian blue staining. (E) <i>Type I collagen</i> expression. (F) <i>Type II collagen</i> expression. (G-I) The distal part of the st. 56 <i>Xenopus</i> limb bud. (G) HE and Alcian blue staining. (H) <i>Type I collagen</i> expression. (I) <i>Type II collagen</i> expression. A-C are shown at same magnification. D-I are at same magnification. Scale bars in A, B insert, D are 500 μm, 200 μm, 100 μm, respectively. Arrowheads indicate presumed cartilaginous regions.</p

<i>Xenopus</i> ALM blastema cells do not have cartilaginous differentiation capacity.

<p>(A) The scheme of the experiment. (B-E) <i>Xenopus</i> ALM blastema cells were grafted to the bone wound site. (B) HE and Alcian blue staining. B’ is a lower magnification image of B. Black lines indicate bone crack area. (C) Alcian blue staining. The cartilaginous callus was visualized by Alcian blue stain. (D, E) Grafted cells were PKH26-positive (red). PKH26-positive cells were not observed in cartilaginous callus. White arrow heads indicate PKH26-positive cells. (F-I) Deep wound ALM blastema cells were grafted to the bone wound site. (F) HE and Alcian blue staining. (G) Alcian blue staining. (H, I) Grafted cells were observed in the cartilaginous callus. (J-M) Control experiment. Normal blastema cells were grafted to the bone wound site. (J) HE and Alcian blue staining. (K) Alcian blue staining. (L, M) Grafted cells were observed in the cartilaginous callus. B-M are shown at the same magnification. Scale bar in B is 200 μm. Scale bar in B’ is 500 μm.</p

<i>Type I</i> and <i>type II collagen</i> expression patterns in the axolotl blastema.

<p>(A-C) The axolotl blastema at 10 days postamputation. (A) HE and Alcian blue staining. (B) <i>Type I collagen</i> expression was analyzed by <i>in situ</i> hybridization. <i>Type I collagen-</i>expressing cells were observed in the blastema mesenchyme and the proximal bone wound region. (C) There was no detectable <i>type II collagen</i> expression. (D-F) At 20 days postamputation. (D) HE and Alcian blue staining. (E) <i>Type I collagen</i> expression was observed in the dermal layer and the proximal bone wound region. (F) <i>Type II collagen</i> expression was observed in the Alcian blue-positive cartilaginous region. (G-I) At 30 days postamputation. (G) HE and Alcian blue staining. (H) <i>Type I collagen</i> expression was observed in the dermal layer and the proximal bone wound region. (I) <i>Type II collagen</i> expression was observed in the Alcian blue-positive cartilaginous region. A-I are shown at the same magnification. A’, B’, C’, G’, G”, H’, H”, I’ and I” are higher magnification images of A, B, C, G, H and I, respectively and A’-C’, G’-H’ and G”-H” are same magnification, Scale bar in A is 1 mm. Scale bar in A’ is 500 μm. Black bars indicate amputated lines. Black arrowheads indicate <i>type I collagen</i> expression.</p

<i>Type I</i> and <i>type II collagen</i> expression patterns during bone fracture healing in axolotl.

<p>(A-C) The axolotl fracture at 10 days postwounding. (A) HE and Alcian blue staining. (B) <i>Type I collagen-</i>expressing cells were observed at the bone wound site. (C) The <i>type II collagen</i> expression area was smaller than the <i>type I collagen</i> expression area. (D-F) The axolotl fracture at 20 days postwounding. (D) HE and Alcian blue staining. A cartilaginous callus was observed in the fracture plane. (E) <i>Type I collagen</i> expression. (F) <i>Type II collagen</i> expression. (G-I) The fracture at 30 days postwounding. (G) HE and Alcian blue staining. (H) <i>Type I collagen</i> expression. (I) <i>Type II collagen</i> expression. A-F are shown at same magnification. G-I are at same magnification. Scale bars in A and G are 500 μm. Black bars indicate the bone fracture plane.</p

<i>Type I</i> and <i>Type II collagen</i> expression patterns in <i>Xenopus</i> stage 52 and stage 56 limb bud blastemas.

<p>(A-C) On day 10 following zeugopod amputation at st. 52 limb bud. (A) HE and Alcian blue staining. (B) <i>Type I collagen</i> expression. <i>Type I collagen</i> expression was weak in the distal region. (C) <i>Type II collagen</i> expression. (D-F) On day 10 following zeugopod amputation at st. 56 limb bud. (D) HE and Alcian blue staining. (E) <i>Type I collagen</i> expression. <i>Type I collagen</i> expression was observed throughout the entire mesenchymal region. (F) <i>Type II collagen</i> expression. A-C are shown at the same magnification. D-F are shown at the same magnification. A’-F’ are higher magnification images of A-F, respectively. Scale bars in A, D, C’, D’, are 200 μm, 500 μm, 1 mm, 250 μm, respectively. Black bars indicate amputated planes. Arrowheads indicate estimated cartilage forming areas.</p

<i>Type I</i> and <i>type II collagen</i> expression patterns of cartilage.

<p>* Only in early stage.</p><p><i>Type I</i> and <i>type II collagen</i> expression patterns of cartilage.</p

Identification and Functional Analysis of Delta-9 Desaturase, a Key Enzyme in PUFA Synthesis, Isolated from the Oleaginous Diatom <i>Fistulifera</i>

<div><p>Oleaginous microalgae are one of the promising resource of nonedible biodiesel fuel (BDF) feed stock alternatives. Now a challenge task is the decrease of the long-chain polyunsaturated fatty acids (PUFAs) content affecting on the BDF oxidative stability by using gene manipulation techniques. However, only the limited knowledge has been available concerning the fatty acid and PUFA synthesis pathways in microalgae. Especially, the function of Δ9 desaturase, which is a key enzyme in PUFA synthesis pathway, has not been determined in diatom. In this study, 4 <i>Δ⁹ desaturase</i> genes (<i>fD9desA</i>, <i>fD9desB</i>, <i>fD9desC</i> and <i>fD9desD</i>) from the oleaginous diatom <i>Fistulifera</i> were newly isolated and functionally characterized. The putative Δ⁹ acyl-CoA desaturases in the endoplasmic reticulum (ER) showed 3 histidine clusters that are well-conserved motifs in the typical Δ⁹ desaturase. Furthermore, the function of these Δ⁹ desaturases was confirmed in the <i>Saccharomyces cerevisiae ole1</i> gene deletion mutant (<i>Δole1</i>). All the putative Δ⁹ acyl-CoA desaturases showed Δ⁹ desaturation activity for C16∶0 fatty acids; fD9desA and fD9desB also showed desaturation activity for C18∶0 fatty acids. This study represents the first functional analysis of Δ⁹ desaturases from oleaginous microalgae and from diatoms as the first enzyme to introduce a double bond in saturated fatty acids during PUFA synthesis. The findings will provide beneficial insights into applying metabolic engineering processes to suppressing PUFA synthesis in this oleaginous microalgal strain.</p></div