Nakajima K , Tazawa I .

Z01 HD001901-12 Intramural NIH HHS, Z01 HD001901-12 NULL

	Fig. 1. Histological analysis of wild-type and TR-knockout tadpole tails at climax of metamorphosis. (A) Schematic representation of stage-63 tail sampling. A tail was sectioned at one-third of the distance from the tail base as indicated by a red arrow. (B–D) A cross sections of wild-type (B), TRα-knockout (C), and TRβ-knockout (D) stage-63 tadpole tail. Histological images are representative of three individuals examined per panel. (E) A cross section of wild type late stage-62 tadpole tail, which was sectioned about at half of the length. Sections were stained with hematoxylin and eosin. Note that the notochord in TRβ-knockout tail at stage 63 resembles that of the wild type at stage 62, showing the presence of vacuolated cells, i.e., not collapsed, compared to the notochord in the wild type or TRα-knockout tail at stage 63. Abbreviations: M, muscle; NC, notochordal cells; NS, notochord sheath; SC, spinal cord. Scale bars, 0.1 mm.
	Fig. 2. Two methods for isolating notochords. (A) A representative photo of notochord preparation by method-A. The tail skin was cut circumferentially at the junction with the body (the tail base) and the tail was pulled to expose the stretched notochord, which was then isolated by cutting at the two ends of the exposed notochord. Scale bar, 10 mm. (B) A representative photo of notochord preparation by method-B. The skin and muscles of left side were peeled to the end of the tail (white arrow) to expose the notochord (red arrow heads), and notochord was then pulled out. In this case, spinal code was attached to left side that was peeled to the end of the tail (thus not visible in figure). Anterior, left; Dorsal, top. Scale bar, 1 mm.
	Fig. 3. TRβ is highly expressed specifically in the tail notochord at the climax of metamorphosis. The expression levels of TRα (A), TRβ (B) and ratio of TRβ to TRα (C) were determined by RT-PCR in the wild-type stage-60 whole tail (St60), stage-63 whole tail (St63), stage-63 notochord-removed-tail (St63dNC), stage-63 notochord prepared by method-A (noto-A), and method-B (noto-B), respectively. The samples included 5, 5, 8 tails and 38, 15 notochords, respectively. (A, B) The expression levels were shown as the copy number per 100,000 copies of EF1α. Data are expressed as mean ± SE of technical replications (n = 5–6).
	Fig. 4. Preferential high level expression of genes encoding matrix metalloproteinases (MMPs) in the tail notochord at climax of metamorphosis. The expression levels of the ECM-degrading MMP2, MMP9-TH, MMP11, MMP13 and MMP14 were determined by RT-PCR in the stage-60 whole tail (St60), stage-63 whole tail (St63), stage-63 notochord-removed-tail (St63dNC), notochord prepared by method-A (noto-A) and method-B (noto-B), respectively. The fold increase between the samples of St60 and St63, between St63dNC and St63 and between St63dNC and the average of noto-A and noto-B are indicated in the figure. Note all MMPs were upregulated at St63 compared to St60. MMP9-TH and MMP13, and to a lesser extent MMP14, were expressed at much higher levels in the notochord compared to the rest of the tail, leading to significantly lower levels of their mRNAs in St63dNC than that in the whole tail even though notochordal RNA is only a tiny fraction of the total RNA in the tail. The individual sample included 5, 5, 8 tails and 38, 15 notochords, respectively. The expression levels were shown as the copy number relative to EF1α. Data are expressed as mean ± SD of technical replications (n = 3–4).
	Fig. 5. Distinct localizations of MMP9-TH, MMP13 and MMP14 expression in the tail at the climax of metamorphosis. Cross sections of the tail at stage 62 were hybridized with antisense MMP9-TH (A, A′, A″), MMP13 (C, C′, C″) or MMP14 (E, E′, E″) probes or their sense probes (B, B′, D, D′, F, F′). Dark purple deposits indicate the sites of probe binding. Black pigments in some areas, e.g., spinal cord (SC), are melanin (see B, D, F). Note that MMP9-TH mRNA is expressed in the outer sheath cells (OS) and weakly in the connective tissue sheath (CT) surrounding the notochord (NC). MMP13 mRNA is expressed in the outer sheath cells, but not in the connective tissue sheath. MMP14 mRNA is expressed in the connective tissue sheath, fibroblast (FB, E′) and subepidermal fibroblast (SF, E″) but not observed in the outer sheath cells. Boxed areas in the panel A-F are magnified in panel A′-F′, A″, C″ and E″. Scale bars, 0.2 mm (A-F) or 0.05 mm (A′-F′, A″, C″ and F″).
	Supplemental Fig. 1: Histological alteration of the notochord under in situ hybridization procedures. (A) Schematic representation of notochordal structure as modified from (Corallo et al. 2015). (B-F) Cross sections of the tail at stage 62 with (C-F) or without (B) treatment under the in situ hybridization procedures. Collagen was stained with 0.1% Sirius red (Sigma) in saturated aqueous solution of picric acid (Sigma) for 1 hour. Then the sections were washed in two changes of 0.5%glacial acetic acid (Junqueira et al. 1979). (C): A section hybridized with the control probe. (D): A section hybridized with MMP9-TH anti sense probe. (E): The same section as in (D) showing nuclei stained with hoechst 33342. (F): The merged image of D and E. Note that the hybridization procedure led to the contraction of the inner notochordal cells (NC) and thus the artificial separation of the connective tissue sheath (CT) and the outer sheath cells (OS), giving rise to a thicker collagenous sheath (CS) in C-F compared to B. NS, notochord sheath, which includes CT and CS. Scale bars, 0.1 mm.

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