Molecular evidence for an activator-inhibitor mechanism in development of embryonic feather branching

Abstract

The developmental basis of morphological complexity remains a central question in developmental and evolutionary biology. Feathers provide a unique system to analyze the development of complex morphological novelties. Here, we describe the interactions between Sonic hedgehog (Shh) and bone morphogenetic protein 2 (Bmp2) signaling during feather barb ridge morphogenesis. We demonstrate that activator-inhibitor models of Shh and Bmp2 signaling in the tubular feather epithelium are sufficient to explain the initial formation of a meristic pattern of barb ridges and the observed variation in barb morphogenesis in chick natal down feathers. Empirical tests support the assumptions of the model that, within the feather ectoderm, Shh (activator) up-regulates its own transcription and that of Bmp2 (inhibitor), whereas Bmp2 signaling down-regulates Shh expression. More complex models incorporating a second activator and dorsal/ventral polarized modification of activator signaling can produce all of the barb morphogenesis patterns observed during the growth of more complex branched pennaceous feathers: new barb ridge formation, helical growth, and barb ridge fusion. An integrated model of feather morphogenesis and evolution suggests that plumulaceous feather structure evolved by the establishment of activator-inhibitor interactions between Shh and Bmp2 signaling in the basal epithelium of the feather germ. Subsequently, pennaceous feather structure evolved through the integration of barb ridge morphogenesis with a second, local inhibitor and a dorsal/ventral signal gradient within the feather. The model is congruent with paleontological evidence that plumulaceous feathers are primitive to pennaceous feathers.

Fig. 1.

Expression dynamics of Shh and Bmp2 in developing embryonic feather buds of plumulaceous and pennaceous feathers. Shh (A) and Bmp2 (B) are expressed in overlapping regions during the development of the feather bud. (C) Shh expression is diffuse in early buds but becomes refined into longitudinal domains, or stripes, in the marginal plate epithelium at the edge of the forming barbs (white arrows) from the initial diffuse expression (black arrow). (D) In plumulaceous down feathers of the chick, the longitudinal Shh expression domains run parallel as the feather grows in length from the bottom. (E) The simple, tufted, plumulaceous, natal down feather of a chick lacks a rachis. (F and G) Pennaceous feather germs in embryonic ducks exhibit helical growth of barb ridges from the ventral (F) to dorsal (G) surfaces. Ventral bifurcation of Shh stripes (F, arrows) leads to the addition of new barb ridges, whereas dorsal extinction of Shh stripes (G, arrows) produces fusion of barb ridges to create the rachis ridge and branched feather form. (H) A pennaceous natal duck feather has a prominent rachis and planar form.

Fig. 2.

Activator–inhibitor models are sufficient to describe the establishment of barb patterning within embryonic feather buds. (A–D) A time series of rings that show the concentration of activator (solid areas) and inhibitor (lines) in a two-component model (Eqs. 1 and 2) producing a stable equilibrium of activator and inhibitor concentrations (D). (E) Stable activator and inhibitor concentrations for a ring in a two-component model with activator saturation. (F) Simulation of barb patterning in a tubular feather germ by a two-component model showing the establishment of a stable number and position of stripes of peak activator concentration from random initial concentrations. (G) Simulation of barb patterning in a feather germ with a growing circumference by using a two-component model with local activator saturation. These conditions create opportunities for initiation of new activator peaks and bifurcation of existing peaks. (H) Simulation of barb patterning in a tubular feather germ by using a three-component model (Eqs. 1–3) and uniform background activator production (circle at top). The stripes of activator concentration grow helically like traveling waves because of an additional, local inhibitor. Positional instability creates opportunities for activator stripe bifurcation, fusion, and cessation, but they are not localized to any particular position in the tube. (I and J) Ventral (I) and dorsal (J) views of a simulation of barb patterning in a tubular feather germ using a three-component model with ventral decrease and dorsal increase in the background level of activator production (b_b in Eq. 1, circles at top). Lowered inhibition of activator autocatalysis causes activator saturation level and leads to stripe branching on the ventral side. The elevated levels of inhibition lead to an extinction of activation peaks as they reach the dorsal side.

Fig. 3.

Effects of forced expression of molecular mediators of Shh and Bmp signaling in feather germs by using localized RCAS infections of the ectoderm during outgrowth and formation of barb ridge pattern. (A–D) Foci of virus expressing a constitutively active BMP receptor 1 (red, caBMPR1) showed a cell autonomous down-regulation of endogenous Shh expression (blue) in placode (A) and early bud stages (B) and during barb specification (C and D). Effect of the virus expression on Shh expression is also seen far from the site of infection (C). (E–H) Virus expressing Shh (blue, RCAS-SHH) caused up-regulation of endogenous Shh expression (red) in early feather buds (E) and later feather filaments (F-H). This effect was seen far from the site of ectopic expression, indicating the long-distance signaling capacity of Shh in the feather epithelium. (I and J) Infection of RCAS-SHH up-regulated endogenous Bmp2 expression (red) in small feather buds (gray outline). Bmp2 expression was detected only near infection sites. (K) Effect of control RCAS virus (blue) on endogenous Shh expression (red). Normal Shh and Bmp2 expression in E-H is present, but the red INT/NBT precipitate is not as sensitive or stable as BCIP/NBT and is diminished in the two-color procedure.