Extended exposure to Sonic hedgehog is required for patterning the posterior digits of the vertebrate limb

Abstract

Sonic hedgehog (Shh) is a key signal in establishing different digit fates along the anterior-posterior axis of the vertebrate limb bud. Although the anterior digits appear to be specified by differential concentrations of Shh in a traditional, morphogen-like response, recent studies have suggested that posterior digits are specified by an extended time of exposure to Shh rather than, or in addition to, a threshold concentration of Shh. This model for digit patterning depends upon continued Shh signaling in the posterior limb through mid-to-late bud stages. We find that cyclopamine, a potent antagonist of Shh signaling, can down-regulate hedgehog target genes in the posterior limb throughout the time Shh is expressed, indicating that continued active Shh signaling indeed takes place. To further explore the relative roles of time and concentration of Shh during limb development, we carried out two additional series of experiments. To test the effect of limiting the time, but not the amount of Shh produced, we treated chick embryos with the hedgehog antagonist cyclopamine at various stages of limb development. We find that short exposures to Shh result in specification of only the most anterior digits and that more posterior digits are specified sequentially with increasing times of uninterrupted Shh activity. To test the effect of limiting the level of Shh produced, but not the time of exposure, we genetically modified Shh production in mice. As previously shown, reducing both the concentration of Shh produced and the duration of Shh exposure results in a loss of posterior digits. We find that maintaining a low level of Shh production throughout the normal time frame of ZPA signaling results in a near complete restoration of the posterior-most digits. These data are consistent with, and lend additional support to, the model that concentration of Shh seen and duration of exposure both contribute to the dose-dependent specification of digit identities, but for the posterior-most digits the temporal component is the more critical parameter.

Figure 1

Proposed model of digit specification. The proposed model is presented here in the context of mouse limb bud development. Digit 1 arises independent of Shh signaling while digit 2 is specified by early diffusion of Shh protein from the posterior margin. Cells fated to give rise to digits 3, 4 and 5 all receive high level autocrine Shh signaling, and differential specification is primarily dependent on the length of time cells are exposed to high level Shh signaling before proliferation/posterior mesenchymal expansion forces cells outside of the zone competent to express Shh.

Figure 2

The posterior forelimb continues to respond to Shh signaling at late stages. Shh, Ptc1, and Ptc2 are all expressed in the posterior limb at stage 21 (A,B,C) and stage 24 (E,F,G). Gli1, though expressed in the very posterior limb at stage 21 (D), is downregulated in the posterior at stage 24 (H). This tissue is still sensitive to Shh signaling at late stages as shown by the downregulation of Ptc1 and Ptc2 12 hours after cyclopamine treatment (J,K). Gli1 is also downregulated (L). Shh continues to be expressed (I) as does the AER domain of Ptc2 expression (K), showing that the effect is not nonspecific. Extended exposure to a Shh bead downregulates Gli1 (M).

Figure 3

Cyclopamine treatment leads to downregulation of Shh targets in the forelimb. Within two hours, Ptc1 is downregulated in treated forelimbs (B) when compared to control forelimbs (A), and its expression ceases within four hours of cyclopamine treatment (C). Gli1 and Bmp2 are downregulated within eight hours (D,F) compared to control forelimbs (E,G). On the other hand, Gli2 expression expands into the posterior forelimb within eight hours of treatment (H) when compared to controls (I).

Figure 4

Shorter exposure to Shh signaling causes the loss or anteriorization of posterior skeletal elements. Digit identities were determined by position and counting phalanges. Control forelimbs (A) and hindlimbs (B) treated with carrier are morphologically indistinguishable from wildtype. Treating with cyclopamine at E3 frequently leads to the loss of the ulna and the reduction of the autopod to carpus and two digit-like elements in the forelimb (C), and the loss of the fibula and digit 4 in the hindlimb (D). Digits 1 - 3 have only two phalanges each, meaning that digits 2 and 3 are anteriorized (D). Treating with cyclopamine at E3.5 frequently leads to the loss of digit 4 in the forelimb and hindlimb (E,F), as well as the reduction of digit 3 in the hindlimb to three phalanges (F). Treating with cyclopamine at E4 leads to a partial digit 4 in the forelimb (G), and to a hindlimb digit 4 with only four phalanges (H). All images were acquired at the same magnification and cropped to 1400×700 pixels. Scale bar represent 1 mm, and is approximated from an image of an identically staged limb taken under equivalent magnification.

Figure 5

A conditional knockout of Shh by the Shhgfpcre causes a continuous low level of Shh signaling throughout limb development. Shh expression expands after eight hours of cyclopamine treatment (A) compared to control forelimbs (B) due to Shh autoregulation. Likewise, a greater amount of the forelimb mesenchyme has attempted to upregulate Shh at E12.5 in Shh nulls (C) than in wildtype (D). At E10.5, Shhgfpcre/Shh^c forelimbs (F) have reduced levels of Shh, compared to wildtype forelimbs (E). At E11.5 Shh is still expressed, albeit at low levels (arrowhead), in Shhgfpcre/Shh^c forelimbs (H) in a domain proximal and anterior to the Shh domain in wildtype forelimbs (G). Shh signaling, as indicated by Ptc1 expression, is significantly reduced in E10.5 Shhgfpcre/Shh^c forelimbs (J) compared to wildtype forelimbs (I). At E11.5, only extremely faint Ptc expression can be observed in E11.5 Shhgfpcre/Shh^c forelimbs (K) compared to wildtype forelimbs (L).

Figure 6

Extended exposure to low levels of Shh leads to posterior digit specification in Shhgfpcre/Shh^c mice. A-C upper panel whole limbs (scale bar represents 1 mm); A-C lower panel (scale bar represents 250 μm) allows analysis of carpal/metacarpal element morphology. Whereas E18.5 wildtype forelimbs have all five digits (A), Shhgfpcre/Shh^c mice only have digits 1-3-4-5 (B), and Prx1Cre;Shh^c/c mice only have digits 1-2-3 (C). Fgf4 expression becomes posteriorly restricted in Shhgfpcre/Shh^c forelimbs (D) when compared to wildtype forelimbs (E). The Shh descendents still make up digits 4 and 5, and part of digit 3 in the Shhgfpcre/Shh^c mice (F) as in wildtype mice (G).

Figure 7

Proposed model of digit specification incorporating mouse mutant analysis. Digit specification in wildtype (A) mouse limb buds as previously detailed (see Figure 1). In Prx1-Cre mutant limb buds (B), early diffusion of Shh allows specification of digit 2 while in the posterior, only digit 3 can be specified before Cre activity abolishes all Shh signaling by E11.0. In contrast, continuous but low level Shh signaling observed in Shh-Cre limb buds (C) is not sufficient to specify digit 2; Posterior digits are however specified with time despite significant reduction of Shh levels. Together, mouse mutant analysis supports the proposed model of digit specification whereby digit 2 is most sensitive to the concentration of Shh while for posterior digit specification, the length of Shh exposure is the more critical parameter.