Initiation of proximal-distal patterning in the vertebrate limb by signals and growth

Abstract

Two broad classes of models have been proposed to explain the patterning of the proximal-distal axis of the vertebrate limb (from the shoulder to the digit tips). Differentiating between them, we demonstrate that early limb mesenchyme in the chick is initially maintained in a state capable of generating all limb segments through exposure to a combination of proximal and distal signals. As the limb bud grows, the proximal limb is established through continued exposure to flank-derived signal(s), whereas the developmental program determining the medial and distal segments is initiated in domains that grow beyond proximal influence. In addition, the system we have developed, combining in vitro and in vivo culture, opens the door to a new level of analysis of patterning mechanisms in the limb.

Fig. 1

Wnt3a, FGF8, and RA act together to maintain markers of early limb mesenchyme in culture. Dissociated fresh HH18 distal limb bud cells were cultured with serum only (serum), serum + Wnt3a and FGF8 (WF), or serum + Wnt3a, FGF8, and RA (WFR) for increasing amounts of time. (A) Sox9, (B) Meis1, (C) Hoxa11, and (D) Hoxa13 expression levels were measured by quantitative PCR and normalized to β-actin expression.

Fig. 2

Expression of Meis1, Hoxa11, and Hoxa13 delineate segmental domains in recombinant limbs. Whole-mount in situ hybridization with Meis1, Hoxa11, and Hoxa13 probes 72 hours after grafting. (A to C) Recombinants using freshly dissociated HH18 hindlimb cells. (D to F) Recombinants using HH18 hindlimb cells cultured for 36 hours in Wnt3a and FGF8. (G to I) Recombinants using HH18 hindlimb cells cultured for 36 hours in the presence of Wnt3a, FGF8, and RA. Scale bars: 500 µm in (A) to (C) and 800 µm in (D) to (I).

Fig. 3

Wnt3a, FGF8, and RA together maintain the potential of cells to form the complete PD axis. (A) Freshly dissociated HH18 hindlimb cells formed three distinct limb segments 14 days after grafting. Although not shown, proximal and middle segments were covered with feathers, and digits with terminal claws were covered by scales. (C and D) Cells cultured for 36 hours in the presence of Wnt3a and FGF8 (WF) lost the ability to form all but a single scale-covered digit extending from a cartilage nodule embedded in the flank (n = 25 out of 28 with one segment). (B and E to H) Cells cultured for 36 hours in Wnt3a, FGF8, and RA (WFR) formed an elongated feather-covered proximal segment, a short feather-covered middle segment, and a scale-covered digit with a terminal claw (n = 13 out of 24 with three segments). F, femur; T/F, tibia and fibula; M, metatarsal; D, digits. Scale bars: 5 mm in (A) and (E), 2.5 mm in (B) and (C), 1.6 mm in (D), and 1 mm in (F) to (H).

Fig. 4

PD potential is restricted by time spent out of the influence of RA. (A) Recombinants made from freshly dissociated distal HH24 hindlimb cells. (B) Recombinants of distal HH24 cells cultured for 36 hours in Wnt3a, FGF8, and RA (WFR) (n = 8). (C and D) Recombinants of HH18 cells cultured for 18 hours in the presence of all three factors followed by 18 hours with Wnt3a and FGF8 (WF) resembled recombinants grafted immediately after culture in Wnt3a and FGF8 for 18 hours (C) n = 28; (D) n = 12. (E and F) HH18 cells cultured for 12 hours in all three factors followed by 24 hours in Wnt3a and FGF8 formed recombinants that resembled those cultured for 24 hours in the two factors alone (E) n = 12; (F) n = 9. Scale bars: 1.5 mm in (A) and (B) and 1 mm in (C) to (F).