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Fig. 3. The Cyc pro-domain regulates processing, stability and signaling. (A) Schematic representation of Cyc, Sqt, Sqt^pro:Cyc^mat, Cyc^pro:Sqt^mat and Cyc2. Hatched boxes indicate the leader sequences, the Cyc pro-domain is shown in pink, the Sqt pro-domain in dark blue, the Sqt mature domain in pale blue and the Cyc mature domain in mid-blue. RNA encoding the various Nodal-related proteins (5-10 pg) was injected into one-cell at the 64- to 128-cell stage together with a lineage tracer (brown). Range of signaling was examined by in situ hybridization to detect ntl (blue, top panel) and gsc (blue-brown, bottom panel) expression. All in situ panels show the boxed area indicated in the schematic of the embryo shown in the left. Expression of ntl is seen in all clones, whereas gsc expression is detected in the clones expressing Sqt, Cyc^pro:Sqt^mat and Cyc2, but not Cyc or Sqt^pro:Cyc^mat. In comparison with Cyc, the range of activity of Cyc2 is markedly expanded, as observed by expression of ntl, and Cyc2 behaves similar to Sqt in inducing gsc. The Sqt^pro:Cyc^mat fusion behaves like Cyc in the expression of both ntl and gsc, whereas ntl expression shows that Cyc^pro:Sqt^mat has reduced signaling activity compared with Sqt. Expression of gsc is detected in some Cyc^pro:Sqt^mat-injected embryos. All embryos were viewed from the animal pole. Scale bar: 100 µm. (B) Table summarizing ntl and gsc expression in the range of signaling assays. For gsc, the embryos were classified as with or without ectopic expression. The induction of ntl was classified as short range (expression in injected cells to three-cell diameters from injected source), long range (expression in cells that are 4-10 cell diameters from source) or not expressing. The amount of injected mRNA (pg), the total number of embryos examined (N) and the percentage of embryos in each category are indicated. (C) Supernatants from HEK293T cells expressing Myc-tagged Sqt, Cyc^pro:Sqt^mat, Sqt^pro:Cyc^mat, Cyc and Cyc2 proteins. Both unprocessed (46 kDa) and processed (17 kDa) forms of Sqt are detected. The Cyc precursor is not detected, whereas substantial amounts of the Sqt^pro:Cyc^mat precursor (45 kDa) are detected. For Cyc^pro:Sqt^mat, no precursor is detected. Very low levels of processed Cyc^mat (16 kDa) is detected from supernatants of cell expressing Cyc, and marginally higher amounts of processed Cyc is detected in the Cyc2 lane (16 kDa). The samples were electrophoresed on a SDS-PAGE gel; lane 6 (Cyc2) is from a different part of the same gel. Expression of the transfection control GFP in cell lysates is shown in the lower panel.

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