Fig. 3.

Collar cell descendants contribute to Fgf10⁺ infundibulum. (A) Boxed region indicates dissection of Fgf3⁺ collar zone cells. (B-D) Immediately after isolation, collar explants are Fgf3⁺ Fgf10⁻ (B,C) but are Fgf10⁺ after 24 hours in culture (D). (E-G) After isolation (E), prospective p/v infundibular cells are Fgf3⁻ (F) and Fgf10⁺ (G). (H) Model for infundibular development. p/v infundibulum (dark green) arises from ventral midline floor plate cells that remain in a midline position. Fgf3⁺ SOX3⁺ collar progenitors (red) can give rise to descendants that populate the a/d infundibulum (light green). Arrows indicate flow of collar descendants to form a/d infundibulum. (I-L) Ventral wholemount (I-K) or thick coronal section (L) views of infundibulum/collar region after collar cells from a Roslin-GFP embryo (head shown in inset, I) were transplanted adjacent to endogenous collar cells in wild-type (wt) embryos, shown schematically (I) or analysed after successive time points (J-L). Immediately after transplantation, GFP⁺ SOX3⁺ collar cells lie close to the endogenous SOX3⁺ collar (J). Analysis of a similar graft reveals that GFP⁺ cells stream medioventrally from the transplant by 24 hours (K, dotted white line indicates the endogenous collar region). The same transplant, analysed at 72 hours (L), reveals more migrating GFP⁺ cells. Some SOX3⁺ collar cells remain in situ; migrating cells downregulate SOX3. (M) Thick coronal section view after transplantation of E4.5 GFP⁺ prospective p/v cells. After 72 hours, transplanted cells remain in situ and do not cross the endogenous SOX3⁺ collar (outlined). (N-P) High-magnification view of L (N) and serial adjacent section analysed for Fgf10 (O). Arrowhead in N points to some SOX3⁺ collar cells that remain in situ. GFP⁺ cells migrate into the Fgf10⁺ infundibular region and express Fgf10 (Pi,Pii). Inset in Pii shows high-magnification view of a GFP⁺ cell that has upregulated Fgf10. (Q) Heterochronic transplant from E10 GFP⁺ to E4.5 wt embryo analysed after 72 hours (thick coronal section). Some GFP⁺ SOX3⁺ collar cells remain in situ in the endogenous SOX3⁺ collar (outlined), whereas other GFP⁺ cells migrate medioventrally, downregulating SOX3. (R) Model showing that collar cell descendants contribute to a/d infundibulum and upregulate Fgf10. Top, yellow circle represents the transplanted collar zone. Bottom, see H. A-P, anterior to posterior. Scale bars: 100 μm in A,E; 25 μm in B-D,F,G; 100 μm in H; 300 μm in I; 50 μm in J-L; 60 μm in N,O; 30 μm in P; 50 μm in M,Q.