Pleiotropic patterning response to activation of Shh signaling in the limb apical ectodermal ridge

Abstract

Sonic hedgehog (Shh) signaling in the limb plays a central role in coordination of limb patterning and outgrowth. Shh expression in the limb is limited to the cells of the zone of polarizing activity (ZPA), located in posterior limb bud mesoderm. Shh is not expressed by limb ectoderm or apical ectodermal ridge (AER), but recent studies suggest a role for AER-Shh signaling in limb patterning. Here, we have examined the effects of activation of Shh signaling in the AER. We find that targeted expression of Shh in the AER activates constitutive Shh signaling throughout the AER and subjacent limb mesoderm, and causes a range of limb patterning defects with progressive severity from mild polydactyly, to polysyndactyly with proximal defects, to severe oligodactyly with phocomelia and partial limb ventralization. Our studies emphasize the importance of control of the timing, level and location of Shh pathway signaling for limb anterior-posterior, proximal-distal, and dorsal-ventral patterning.

Fig. 1. Msx2 sequences used to ectopically express Shh in the AER of transgenic mice

(A) Map of chicken Msx2 gene, (B) Msx2-LacZ reporter construct, and (C) Msx2-Shh transgene construct. (D) Msx2-LacZ founder embryo limbs showing expression of β-galactosidase exclusively in the AER. (E) Msx2-LacZ founder embryo limbs showing expression of β-galactosidase in the AER, and also in the ventral ectoderm. The level of expression is higher than in the embryo shown in (D).

Fig. 2. Limb phenotypes of newborn Msx2-Shh mutant mice

(A, B) Wildtype littermate forelimbs and (D, E) wildtype littermate hindlimbs stained whole mount with Alcian blue and Alizarin red to visualize the normal skeletal element pattern. (C, F) Dermatoglyphic analysis of normal limbs showing (C) carpal pads (carp), digital pads (dp), interdigital pads (idp) and caterpillar pads (catp) on the normal ventral forelimb surface; and (F) tibiotarsal pad (ttp) and cobblestone pads (cobp) on the normal ventral hindlimb surface. (G–L) Mildly affected polydactylous Msx2-Shh forelimbs (G–I) and hindlimbs (J–L) showing ectopic preaxial digits with anterior identity (arrows in H,K), ectopic interdigital pads (arrows in I, L), broadened digit nails and split digit tips (double arrows in I, K), and anterior shift in position of the tibiotarsal pad (arrowhead in L). (M–R) Moderately affected polysyndactylous Msx2-Shh forelimbs (M–O) and hindlimbs (P–R) with multiple indistinguishable ectopic digits with posterior identity (arrows in N, Q) and loss of digit 1, branched digit tips and duplicated nails (double arrows in N, O, Q, R), and missing tibia (arrow in P) accompanied by loss of the tibiotarsal pad and replacement by cobblestone pads (arrowhead in R). (S–Z) Severely affected oligodactylous and phocomelic Msx2-Shh forelimbs (S–U′) and hindlimbs (V–Z) showing shortened limbs with fused and/or malformed digits with posterior identity, fused and unidentifiable carpals and tarsals, short and fused zeugopod (arrow in S), or absent zeugopod and stylopod (arrows in V, W). The arrow in (X) indicates the persistent posterior metatarsal. Footpads are present on the ventral surfaces of the oligodactylous limbs but are deformed and fused (U, U′, Y, Y′). Ventral footpads are present on the dorsal surface of the oligodactylous hindlimbs (arrows in Z). All limbs are ventral views with anterior to the left/top except for the dorsal view of the hindlimb shown in (Z).

Fig. 3. Progression of digit formation in Msx2-Shh mutant mice

Comparison of normal littermate (A–D) and mutant (E–H) Msx2-Shh hindlimbs in individual embryos between E12.5 and E16.5 in polydactylous line 13 stained whole mount with Alcian blue. The distal end of the mutant limb is widened as early as day E12.5 and digit branching and an ectopic digit condensation (arrows) are apparent (E). Complete extra digits, partial extra digits and branched digits are formed in the mutant limbs between days E14.5-E16.5 (arrows). Note also the deformed zeugopod elements in the E14.5-E16.5 mutant hindlimbs (F–H). Anterior is to the right.

Fig. 4. In situ hybridization analysis of chicken Shh (cShh) expression in wildtype and transgenic Msx2-Shh limbs

(A–H) cShh is expressed intensely and in a widespread fashion throughout the AER of most transgenic limbs of polydactylous line 13 (A–D, E, G), while in a few limbs cShh is expressed in a weak or restricted fashion in the AER (F, H). (I–L) Wildtype littermate non-transgenic limbs showing absence of cShh expression in the AER. Anterior is to the left.

Fig. 5. In situ hybridization analysis of mouse Ptch expression (mPtch) in wildtype and transgenic Msx2-Shh limbs

(A, C, E, G) Wildtype day E10.5 line 13 forelimbs (A, C) and hindlimbs (E, G) showing the normal posterior domain of mPtch expression, and little/no expression of mPtch in anterior mesoderm (arrows). (B, D, F, H) Transgenic day E10.5 line 13 forelimbs (B, D) and hindlimbs (F, H) showing that in addition to the normal posterior domain of mPtch expression, mPtch is ectopically expressed in anterior and distal mesoderm of the transgenic limbs (arrows). The domain of mPtch expression is delineated by dotted lines. Anterior is to the left.

Fig. 6. In situ hybridization analysis of murine patterning genes expressed in wildtype and transgenic Msx2-Shh limbs

(A, B) Mouse Shh is expressed in its normal posterior location corresponding to the ZPA in both wildtype (A) and transgenic (B) Msx2-Shh limbs from polydactylous line 13. (C, D) Fgf4 is restricted to the posterior 2/3 of the AER of the wildtype limb (arrow in C), but ectopically expressed in the anterior AER of the transgenic Msx2-Shh limb (arrow in D). (E, F) Msx2 is expressed by the AER of both wildtype (E) and transgenic (F) Msx2-Shh limbs. However, although high levels of Msx2 transcripts are found in the anterior mesoderm of the wildtype limb bud (E), expression of Msx2 in the anterior mesoderm of the transgenic limb bud is severely reduced (arrow in F). (G–J) Hoxd11 is restricted to the posterior mesoderm of wildtype limbs (G, I) but ectopically expressed in the anterior mesoderm of transgenic Msx2-Shh limbs (arrows in H, J). (K,L) Hoxd13 is expressed by the distal and posterior mesoderm of wildtype limb (K) but is ectopically expressed by the anterior mesoderm of the transgenic Msx2-Shh limb (arrow in L). (A–H, K, L are forelimbs; I, J are hindlimbs; A–J are day E10.5; K, L are day E11.5). Anterior is to the left.