Fig. 5. Pseudotime, CellChat, and functional analysis on the growth of short and long feathers.

(A and B) Pseudotime analysis in short and long feathers. Color map indicates diffusion pseudotime or the progression along the epidermal lineage, while the thickness of solid black lines indicates the probability of transition between epidermal cell states. Intermediate layer and TA cells are predicted to transition faster toward the differentiated epidermal states in the short feather, as demonstrated by the higher pseudotime value. (C) The comparison of IGF and FGF pathway interaction strength between short and long feathers. Top: IGF pathway. Bottom: FGF pathway. Red arrow indicates the stronger IGF pathway interaction in long feathers. Green arrow indicates a specific FGF pathway interaction in long feathers. (D and E) Overexpression of IGF2 in the right wing leads to increased feather filament length (E13). (F to H′) Comparison of H&E and PCNA staining between control and IGF2 overexpressed samples. Note the enlarged feather follicle width (F′) and the expanded PCNA expression domain (G′) (red arrow) in the RCAS-IGF2–injected sample on the right wing, compared to the control follicle in the left wing. (I and J) Inhibition of FGF signaling by RCAS-FGFR1-Fc can significantly reduce the feather length (E13). (K to M′) Comparison of H&E and PCNA staining between control and RCAS-FGFR1-Fc–infected samples. Note the reduced feather follicle width (K′) and the reduced number of PCNA-positive cells (L′) (white arrow) in the RCAS-FGFR1-Fc–infected sample from the right wing, compared to the control follicle in the left wing. (N) Summary of activated IGF and FGF pathway activities in short and longer feathers. For [(E) and (J)], five flight feather filaments from the right (experiment) and left (control) wings were measured. ***P < 0.001.