Figure 3. Short Sog protein acts in a dominant negative manner to regulate onset of TGF-β signaling in the embryo.
(A) Full Sog protein and predicted Short Sog protein product based on 3’ RACE sequence, with functional domains shown. SP=signal peptide, CR=Cystine repeat domain, CHRD=Chordin domain.
(B) Full Sog and Short Sog bound to TGF-β ligands Dpp and Scw, along with binding partner Twisted gastrulation (Tsg) and protease Tolloid (Tld) (Shimmi and O’Connor, 2003). Short Sog lacks Tld cleavage sites (red in Sog), and instead contains amino acids from intron-derived sequence (blue; see also Figure S1A).
(C) In situ hybridization in eve stripe 2-short sog embryo, mid NC14, using riboprobe to first exon of sog, which recognizes both short and long forms of sog, and demonstrates ectopic expression of short sog transcript in the eve stripe 2 domain.
(D,E,H,I) FISH colocalization using riboprobes to the TGF-β target gene race (blue) as well as sog (red) at NC14D in wild type embryos (D,E) or eve2-short sog background (H,I). Dorsal (D,H; surface plane) and lateral (E,I; sagittal plane) views show single stripe of race gene expression present dorsally in wildtype embryos (D,E), while in comparison race expression in eve2-short sog background is diminished and excluded from the trunk (H,I). sog expression is only apparent in lateral view of wildtype embryos (E), but eve2-short sog construct supports additional expression in a stripe at the anterior (H and I, see arrowhead).
(F,G,J,K) Expression of race in gd7 NC14C (F,J) and or gastrulating (G,K) embryos from eve stripe 2-short sog background (F,G), eve stripe 2- sog background (J), or native background, not containing any transgene (K).
(L-O) FISH detection using riboprobes to sog (red) or race (blue) coupled with anti-pMad immunostaining (green) in wild type (L, M) or eve stripe 2-short sog (N,O) embryos. Lateral (L,N) and dorsal (M, O) views are shown. Images are confocal single scans of the embryos’ surface at NC14C-D.
(P-W,Y,Z) race expression in yw (P,T), sog ΔNew 3’ UTR embryos (Q,U), sog (R,V), sog P-element (S,W), and sogY506 mutant (Y,Z) embryos. Shown are lateral views of NC14 early stage (P,Q,R,S,Y) or dorsal views of NC14 late stage (T,U,V,W,Z) embryos. Precocious expression of race (Q,R,Y) correlates with loss of short sog activity.
(X) A diagram of the sog locus with sites of genomic manipulation noted. ΔNew 3’ UTR is the CRISPR-Cas9 deletion of the short sog coding sequence derived from the intron. ΔSxl sites is the CRISPR-Cas9 deletion of ~1kb of genomic DNA containing the Sxl binding sites. Green arrowhead indicates the insertion location of sog P-element: P{GSV2}GS51273.
(See also Figure S4)