XB-IMG-83702
Xenbase Image ID: 83702
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Figure 4.
Dynamically Controlled Protein Accumulation in the Chordin-Sizzled Feedback System
(A-E) Endogenous proteins were recovered from extracellular fluid by dissociating embryos in a small volume of cold CMFM (Ca2+/Mg2+-free medium) with 7.5 mM EDTA at 4ºC (5 μl per embryo; 25 μl per lane for loading). In (A) and (B), increase of endogenous Sizzled protein by Chordin depletion (lane 3) is shown by western blot. Sizzled protein in the same number of stage 12 embryos was loaded in each lane. (B) shows protein quantification in (A). The level of Sizzled in the control embryo was defined as 1. (C) through (E) show accumulation of endogenous Chordin and Sizzled proteins during gastrulation. Secreted fractions in the control (C) and sizzled-MO-injected (D) embryos were analyzed. (E) Quantification of protein levels.
(F-H) Stage-dependent Chordin stability was analyzed in the blastocoel injection assay. In (G), the intact form of recombinant Chordin protein was quantified 1.5 hr after blastocoel injection (at stage 9, 10, 11, or 12). (H) shows the line graph presentation of results in (G).
(I) Effects of Sizzled depletion and overexpression on increased stability of recombinant Chordin at stage 11 in the blastocoel injection assay.
(J) Injection of chordin-MO (bottom) increased the stability of recombinant Chordin in the blastocoel injection assay.
(K) Depletion of all four BMPs (lane 5) promoted Chordin degradation in the stage 11 embryo.
(L) The amount of intact V5-tagged Chordin protein (top) was decreased when nontagged chordin (100 pg; lane 5) was coinjected with V5-tagged chordin (25 pg) into a vegetal blastomere of eight-cell embryos (compare with lane 3). β-CN-MO, Sizzled-MO, and Szl mRNA were injected at the two-cell stage. luc/F (bottom, injection control) and dnBMPR mRNA were injected at the eight-cell stage.
(M-P) Effects of MOs for admp and sizzled on DV markers. ADMP depletion moderately expanded the expression of the dorsal axial marker and reduced that of the ventral marker; 100%, n = 20, in (N). Sizzled depletion suppressed the dorsal axial marker and dramatically expanded the ventral marker; 100%, n = 14, in (O). Combined depletion of ADMP and Sizzled caused a phenotype similar to that by Sizzled depletion; in (P) 100%, n = 16, for dramatic sizzled expansion with no substantial shh expansion.
(Q) Schematic of the long-range feedback and accumulation model. Distribution of Chordin and Sizzled along the DV axis is shown in red and blue, respectively. ns, nonspecific band. Image published in: Inomata H et al. (2013) Copyright © 2013. Image reproduced with permission of the Publisher, Elsevier B. V.
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