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

Authors

  • Chi-Kuang Leo Wang,

    1. Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut
    2. Department of Animal Genetics and Transgenic Animal Facility, University of Connecticut, Farmington, Connecticut
    3. Department of Molecular and Cell Biology, University of Connecticut, Farmington, Connecticut
    4. National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan
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  • Mizuyo H. Tsugane,

    1. Department of Anatomy, Sapporo Medical University, Hokkaido, Japan
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  • Victoria Scranton,

    1. Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut
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  • Robert A. Kosher,

    1. Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut
    2. Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, Connecticut
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  • Louis J. Pierro,

    1. National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan
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  • William B. Upholt,

    1. Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut
    2. Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, Connecticut
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  • Caroline N. Dealy

    Corresponding author
    1. Department of Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut
    2. Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, Connecticut
    • Center for Regenerative Medicine and Skeletal Development, Department of Reconstructive Sciences, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030
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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. Developmental Dynamics 240:1289–1302, 2011. © 2011 Wiley-Liss, Inc.

INTRODUCTION

Patterning of the vertebrate embryonic limb along the proximal–distal (PD), anterior–posterior (AP), and dorsal–ventral (DV) axes is controlled by the coordinated activity of three organizing centers (Benazet and Zeller, 2009; Butterfield et al., 2010) and mediated by key signaling pathways using secreted factors belonging to the fibroblast growth factor (FGF), Hh, bone morphogenetic protein (BMP) and Wnt pathways (Duboc and Logan, 2009; Yang, 2009). The apical ectodermal ridge (AER), a thickened distal epithelium, maintains the proliferation of underlying distal mesoderm and promotes PD outgrowth and patterning by means of synthesis and secretion of FGFs (Mariani et al., 2008). The AER also maintains the activity of the zone of polarizing activity (ZPA), a region of posterior mesoderm that controls the number and identity of the structures of the limb along the AP axis. Shh is a secreted factor expressed and synthesized by the cells of the ZPA (Riddle et al., 1993), which specifies the AP identity of the digits (Bastida and Ros, 2008; Benazet and Zeller, 2009). Nonridge dorsal ectoderm, by means of expression of Wnt7a (Dealy et al., 1993; Riddle et al., 1995), controls limb DV patterning, which determines limb flexion and specifies formation of integument derivatives. The processes of limb outgrowth and patterning are linked by means of feedback loops between the AER, the limb mesoderm and ZPA, and the dorsal ectoderm. FGF4 and Wnt7a in AER and dorsal ectoderm act together to maintain Shh expression in the ZPA (Yang and Niswander, 1995), and Shh in turn maintains AER FGF expression (Niswander et al., 1994). Shh produced by the ZPA also regulates expression of the BMP antagonist Gremlin, which is required for continued maintenance of the AER while the limb is growing (Zuniga et al., 1999; Scherz et al., 2004). Thus, Shh signaling plays a central role not only in specification of AP digit identity, but also in coordination of limb outgrowth and PD and DV patterning, and accordingly, studies have emphasized the importance of control of Shh signaling in the limb, and implicate timing, location, and dose as critical modifiers of Shh activity (Harfe et al., 2004; Bastida and Ros, 2008; Towers et al., 2008; Zhu et al., 2008; Yang, 2009).

An additional role for Shh in limb patterning, mediated by the AER and limb ectoderm, has also been suggested (Bell et al., 2005; Bouldin et al., 2010). Shh is not expressed by the AER or limb ectoderm (Riddle et al., 1993; Bell et al., 2005), but Shh protein has been detected (Bell et al., 2005; Bouldin et al., 2010) and the presence of downstream signaling components of the Shh pathway including Ptch, Smo, and Gli1-3 has been demonstrated by immunohistochemistry, in situ hybridization and/or microarray analysis of isolated ectodermal hulls (Bell et al., 2005; Bouldin et al., 2010). Moreover, a functional role for AER–Shh signaling has been demonstrated through targeted loss-of-function of Shh signaling in the AER of transgenic mice (Bouldin et al., 2010), through Cre-mediated deletion of the Shh mediator smo from the AER by means of the Msx2-Cre allele, which expresses Cre recombinase in the AER but not in limb ectoderm (Fu et al., 2007).

The tissue-specific loss-of-function provided by Msx2-Cre-mediated gene deletion has been used to examine patterning roles of various signaling pathways present in the AER including Shh (Bouldin et al., 2010), as well as FGF, MapK, Wnt 3a, and Notch (Lewandoski et al., 2000; Sun et al., 2000, 2002; Barrow et al., 2003; Pan et al., 2004, 2005; Delgado et al., 2008; Mukhopadhyay et al., 2010). In a previous study, we used a complementary, gain-of-function approach, to examine patterning roles of signaling pathways in the AER (Wang et al., 2004), in which an AER-specific promoter element of the Msx2 gene is used to target expression of genes of interest throughout the AER in transgenic mice (Liu et al., 1994; Sumoy et al., 1995). Here we have used this approach to express Shh in the AER to investigate the gain-of-function effects of Shh-AER signaling on limb patterning. We find that Shh expressed by the AER activates constitutive Shh signaling in 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 AP, PD, and DV patterning.

RESULTS

Shh was expressed in the AER of transgenic mice using a 5.5-kb sequence of the chicken Msx2 promoter (Fig. 1) which exhibits the same temporal and spatial pattern of activity as that previously described for the mouse AER-specific Msx2 promoter (Kimmel et al., 2000; Sun et al., 2000; Barrow et al., 2003). LacZ expressed by this construct is detectable by β-gal staining at embryonic day (E) 9.5 in the limb ventral ectoderm that comprises the prospective AER of the hindlimb, and by the ventral ectoderm and nascent AER of the forelimb (not shown). By E11.5, lacZ reporter expression in the majority of the limbs of Msx2-LacZ embryos (19/22 or 86%) is localized nearly exclusively to the AER (Fig. 1), accompanied by a very low level of expression in the limb ventral ectoderm represented by the presence of one or more LacZ positive cells. A few (3/22 or 14%) of Msx2-LacZ embryos had high levels of expression in the ventral ectoderm, in addition to the LacZ expression in the AER (Fig. 1).

Figure 1.

Msx2 sequences used to ectopically express Shh in the AER of transgenic mice. A–C: Map of chicken Msx2 gene (A), Msx2-LacZ reporter construct (B), and Msx2-Shh transgene construct (C). D: Msx2-LacZ founder embryo limbs showing expression of β-galactosidase exclusively in the apical ectodermal ridge (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.

LacZ expressed by this 5.5-kb Msx2 sequence is also expressed in the hindbrain ectoderm in the region of the developing choroid plexus and in the genital tubercle (Sumoy et al., 1995). Hindbrain defects were present in 80% of the Msx2-Shh transgenic founders and varied from bruises and soft spots underneath the skin of the hindbrain region to open skulls with the brain tissues exposed (Supp. Fig. S1, which is available online). The high rate of central nervous system (CNS) defects led to a high rate of neonatal death of the founders (15/22 or 68%). The fertility rate was also reduced in founder animals and in offspring produced in transgenic lines generated from surviving founders.

Whole-mount Alcian blue and Alizarin red staining revealed that patterning was disrupted in the limbs of Msx2-Shh mutant founder animals and offspring (Fig. 2; Table 1). Overall, the limb phenotypes ranged in progressive severity from a mild polydactylous phenotype with up to three extra preaxial digits with anterior identity (Fig. 2G–L); to a moderate polysyndactylous phenotype possessing six to seven similar, often branched digits and no identifiable digit 1, and accompanied in hindlimbs by proximal defects (Fig. 2M–R); to a severe oligodactylous and phocomelic phenotype with two to three missing digits and severe disruption and/or loss of stylopod and zeugopod elements (Fig. 2S–Z). Anterior structures were primarily affected with little affect on posterior structures (see details below). Approximately 40% of the founder animals had one or more limbs with the mildest polydactylous phenotype, approximately 63% had limbs with the more severe moderate polysyndactylous phenotype, and approximately 30% of the founders had one or more very severely affected oligodactylous/phocomelic limbs (Table 1). Within individual animals, Msx2-Shh mutant hindlimbs were always more severely affected than forelimbs, and overall, a greater number of hindlimbs were observed with oligodactyly compared with forelimbs (Table 1). Disruption of patterning of structures proximal to the autopod was also more common in hindlimbs than in forelimbs (Table 1; Fig. 2). A low frequency of limbs (approximately 20% overall) with no overt phenotype was observed. The presence of unaffected limbs was typically accompanied by additional limbs with the mildest polydactylous phenotype. Msx2-Shh animals possessing limbs with the severest oligodactylous/phocomelic phenotype almost never had any normal limbs (Table 1).

Figure 2.

Limb phenotypes of newborn Msx2-Shh mutant mice. A,B,D,E: Wild-type littermate forelimbs (A,B) and wild-type littermate hindlimbs (D,E) 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′). Z: Ventral footpads are present on the dorsal surface of the oligodactylous hindlimbs (arrows). All limbs are ventral views with anterior to the left/top except for the dorsal view of the hindlimb shown in Z.

Table 1. Distribution of Phenotypes in Msx2-Shh Limbs
 No. analyzedNormalMild polydactylyModerate synpolydactylyOligodactyly/ Phocomelia
Forelimbs
 Founders400%37%48%15%
 Line 1311425%75%0%0%
 Line 18460%28%52%20%
 Line 780%25%75%0%
 Line 8425%75%0%0%
 Line 12244%96%0%0%
Hindlimbs
 Founders430%27%41%32%
 Line 1311415%78%6%1%
 Line 18450%20%26%54%
 Line 780%25%0%75%
 Line 8450%25%25%0%
 Line 12248%92%0%0%

Mildly affected limbs were characterized primarily by preaxial polydactyly, with five to eight digits (Fig. 2G–L). In wild-type limbs, the anterior-most digit (digit 1) is the shortest, and contains only two phalanges (Fig. 2A–F). In contrast, the anterior-most digit of the polydactylous mutants frequently had three phalanges, as are found in normal posterior digits 2–5. The three most posterior digits of the polydactylous limbs appeared normal. No proximal patterning malformations were observed in the mildly affected polydactylous mutants.

Moderately affected limbs were characterized by polysyndactyly and had five to seven digits in the forelimb and four to six digits in the hindlimb, with all or most digits lacking interdigital spaces (Fig. 2M–R). In all cases examined there was no identifiable digit 1. The rest of the digits which were posterior in identity based on their Alizarin red staining and/or presence of three phalanges, appeared similar to each other and often possessed duplicated distal phalanges (Fig. 2N,K) resulting in wide or split nails (Fig. 2N,K,Q) or supernumerary rudimentary digit tips (Fig. 2K). Proximal patterning malformations were observed in the moderately affected polysyndactylous mutants, which were limited to the hindlimbs and were typified by an absent tibia (Fig. 2P). Loss of the tibia is consistent with the loss of more distal anterior structures in the polydactylous Msx2-Shh limbs.

Severely affected Msx2-Shh limbs possessed oligodactyly, with one to three missing digits. In addition, both forelimbs and hindlimbs had severe proximal patterning defects. In the forelimb, the carpals were fused, the radius/ulna were fused and shortened, and the humerus was missing (Fig. 2S,T). Based on their staining with Alizarin red, the remaining digits in the oligodactylous forelimb appear to be middle and posterior digits (compare Fig. 2T with B). In the hindlimb, the normal elements of the zeugopod and stylopod either were missing altogether or replaced by discontinuous unidentifiable cartilage rudiments (Fig. 2V,W). In one case, the pubis was also affected (Fig. 2W). Even though stylopod and zeugopod patterning in the mutant oligodactylous limbs was severely disrupted, intact autopod structures including phalanges and metatarsals could still be identified in some severely affected forelimbs and hindlimbs, indicating a phocomelic phenotype (Fig. 2T,X). Among the few remaining metatarsals of the severely affected hindlimbs, a posterior metatarsal could still be seen in all three stained founders with this limb reduction phenotype (e.g., arrow in Fig. 2X), indicating persistence of posterior structures but loss of anterior ones in the Msx2-Shh hindlimbs.

Dermatoglyphic analysis of the external features of the skin of the mildly polydactylous and moderately polysyndactylous Msx2-Shh forelimbs (Fig. 2C,I,O) and hindlimbs (Fig. 2F,L,R) confirmed the normal appearance of posterior digits and the anterior identity of extra preaxial digits, which possessed appropriate digital and caterpillar pads, and were accompanied by appropriate corresponding ectopic interdigital pads. Widened and/or duplicated nails were also observed on several of the mutant forelimb digits (Fig. 2O,R). Polydactylous Msx2-Shh hindlimbs also possessed these features (Fig. 2L,R). Polydactylous Msx2-Shh forelimbs also possessed normal appearing carpal pads, consistent with lack of disruption of proximal patterning in these limbs. However, in Msx2-Shh polydactylous hindlimbs, in which proximal patterning is disrupted, dermatoglyphic analysis revealed altered position and/or loss of the tibial tarsal pad consistent with absence of the tibia (Fig. 2L,R). Dermatoglyphic analysis of severely affected oligodactylous forelimbs (Fig. 2U,U′) revealed that carpal and interdigit pads were present, but malformed and fused, consistent with malformation and fusion of the autopod elements. In the oligodactylous hindlimbs (Y,Y′,Z), ventral cobblestone pads and interdigit pads were present on the autopod but difficult to identify, and tibial tarsal pads were absent, consistent with the missing proximal elements. In addition, ventral footpads were observed on the dorsal surface of each of the three mutant oligodactylous hindlimbs examined (e.g., Fig. 2Z). The number of ectopic footpads ranged from one to three.

The progression of polydactyly in limbs from a line of Msx2-Shh transgenic animals with the relatively mild polydactylous phenotype (line 13, as shown in Fig. 2G–L) was examined by whole-mount Alcian blue staining (Fig. 3). As early as E12.5, in comparison to normal limbs (Fig. 3A), the autopods of the mutant limbs were visibly widened, and branching of the distal digits and formation of ectopic preaxial cartilage condensations was apparent (Fig. 3E). By E14.5–E16.5, compared with normal limbs (Fig. 3B–D), obvious branched and/or duplicated partial or complete preaxial digits were observed in mutant Msx2-Shh limbs, and frequently the anterior most digit was thinner and less developed than the other digits (Fig. 3F–H).

Figure 3.

Progression of digit formation in Msx2-Shh mutant mice. A–H: Comparison of normal littermate (A–D) and mutant (E–H) Msx2-Shh hindlimbs in individual embryos between embryonic day (E) 12.5 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 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 and E16.5 (arrows). F–H: Note also the deformed zeugopod elements in the E14.5–E16.5 mutant hindlimbs. Anterior is to the right.

In situ hybridization with a chicken Shh probe was used to examine expression of ectopic chicken Shh (cShh) mRNA in the AER of E10.5 and E11.5 transgenic polydactylous line 13 Msx2-Shh forelimbs and hindlimbs and wild-type littermate limbs (Fig. 4). The frequency and distribution of cShh expression in the limbs is shown in Table 2. The majority of the transgenic forelimbs and hindlimbs expressed intense, uniform expression of cShh throughout the entire AER (Fig. 4A–F). The percentage of limbs with this intense and widespread ectopic cShh expression was 16/21 or 76% (Table 2). In a minority of forelimbs and hindlimbs the expression of cShh expression in the AER was weak or interrupted by nonexpressing cells, or was restricted in domain (e.g., Fig. 4G,H). The percentage of limbs with weak or restricted ectopic cShh AER expression was 5/21 or 24% (Table 2). This frequency compares well with the low frequency of unaffected limbs in this line, which was 20% overall (Table 1). No ventral limb ectoderm expression of cShh was detected (not shown). All transgenic limbs exhibited ectopic AER cShh expression (Table 2), while as expected, cShh was not detected in the limbs of littermate nontransgenic animals (Fig. 4I–L), confirming specificity of the cShh probe.

Figure 4.

In situ hybridization analysis of chicken Shh (cShh) expression in wild-type and transgenic Msx2-Shh limbs. A–H: cShh is expressed intensely and in a widespread manner throughout the apical ectodermal ridge (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 manner in the AER (F,H). I–L: Wild-type littermate nontransgenic limbs showing absence of cShh expression in the AER. Anterior is to the left.

Table 2. Distribution of Pattern and Intensity of cShh and mPtch Expression in Msx2-Shh Limbs (Line 13)a
Transgenic cShh expressionNo. analyzedNo AER expressionWeak or restricted AER expressionIntense and widespread AER expression
  • a

    AER, apical ectodermal ridge; E, embryonic day.

E10.5 forelimb50%20%80%
E10.5 hindlimb50%40%60%
E11.5 forelimb50%20%80%
E11.5 hindlimb60%17%83%
mPtch expressionNo. analyzedPosterior expressionWeak or no ectopic anterior expressionStrong ectopic anterior expression
E10.5 forelimb8100%13%87%
E10.5 hindlimb4100%25%75%

Ptch is a Shh-responsive gene which is a sensitive indicator of the presence of Shh signaling. In situ hybridization was used to examine expression of mouse Ptch (mPtch) in E10.5 transgenic and wild-type line 13 Msx2-Shh forelimbs and hindlimbs (Fig. 5). In wild-type limbs (Fig. 5A,C,E,G), mPtch is expressed in a mesodermal domain encompassing the ZPA and extending throughout the posterior mesoderm, and is not expressed in the anterior mesoderm. In contrast, in transgenic Msx2-Shh limbs, mPtch is expressed not only in its normal posterior domain, but in addition is expressed ectopically in a broad distal and anterior domain extending from the posterior mesoderm to the anterior mesoderm throughout the mesenchyme under the AER (Fig. 5B,D,F,H), indicating widespread activation of Shh signaling throughout the mutant mesoderm. The distribution of mPtch expression in transgenic Msx2-Shh limbs is shown in Table 2. We found that 83% (10/12) transgenic forelimbs and hindlimbs express mPtch in an ectopic anterior distal domain. A low frequency (2/12 or 17%) of transgenic limbs exhibited little or no ectopic anterior mPtch expression. This frequency compares well with the low frequency of unaffected limbs in this line, which was 20% overall (Table 1). Importantly, all transgenic limbs examined exhibited a normal posterior domain of mPtch expression (Table 2).

Figure 5.

In situ hybridization analysis of mouse Ptch expression (mPtch) in wild-type and transgenic Msx2-Shh limbs. A,C,E,G: Wild-type embryonic day (E) 10.5 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 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.

Shh, Fgf4, Msx2, Hoxd11, and Hoxd13 are genes which have localized patterns of expression in the developing limb which are modified in polydactylous conditions. In situ hybridization at E10.5 was used to examine the patterns of expression of these genes in line 13 transgenic polydactylous Msx2-Shh and nontransgenic wild-type littermate limbs (Fig. 6). Mouse Shh (mShh) expression was limited to the posterior mesoderm (ZPA) in both wild-type and mutant limbs, and the size and intensity of the domain of mShh expression did not appear to be altered in mutant limbs compared with wild-type limbs (Fig. 6A,B). Fgf4 is expressed only in the posterior 2/3 of the AER of normal limb buds (Fig. 6C), but in the limbs of the transgenic Msx2-Shh embryos, mFgf4 transcripts were detected in a domain which extended nearly to the anterior extent of the limb periphery (Fig. 6D). Msx2 is expressed in the anterior mesoderm of wild-type limb buds (Fig. 6E) but in the mutant limbs the normal domain of Msx2 expression in the anterior mesoderm was suppressed (Fig. 5F). The absence of Msx2 transcripts is consistent with our previous studies demonstrating rapid cessation of Msx2 expression in the anterior mesoderm of the chick limb bud following a ZPA graft (Coelho et al., 1992). mMsx2 transcripts were detected in the AERs of transgenic limbs in a pattern identical to that seen in normal limbs (data not shown). Hoxd11, which is downstream of Shh and is normally expressed only in posterior mesoderm (as shown in Fig. 6G,I), was ectopically expressed in the anterior mesoderm of the mutant limbs (Fig. 5H,J). Similarly, an ectopic domain of mHoxd13 was also present in the anterior mesoderm of the Msx2-Shh limbs (Fig. 6K vs. L).

Figure 6.

In situ hybridization analysis of murine patterning genes expressed in wild-type and transgenic Msx2-Shh limbs. A,B: Mouse Shh is expressed in its normal posterior location corresponding to the zone of polarizing activity (ZPA) in both wild-type (A) and transgenic (B) Msx2-Shh limbs from polydactylous line 13C,D: Fgf4 is restricted to the posterior 2/3 of the apical ectodermal ridge (AER) of the wild-type 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 wild-type (E) and transgenic (F) Msx2-Shh limbs. However, although high levels of Msx2 transcripts are found in the anterior mesoderm of the wild-type 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 wild-type 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 wild-type 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 embryonic day (E) 10.5; K,L are E11.5. Anterior is to the left.

DISCUSSION

The limbs of Msx2-Shh mice exhibit a range of phenotypes with varying severity, progressing from mild polydactyly, to moderately severe polysyndactyly with proximal defects, to oligodactyly with severe proximal defects and phocomelia. This range of phenotypic severity likely reflects differences in the timing, dosage, and/or location of Shh activation. Recently, a functional role for AER–Shh signaling has been demonstrated through targeted loss-of-function of Shh signaling in the AER of transgenic mice, through Cre-mediated deletion of the Shh mediator smo from the AER by means of the Msx2-Cre allele (Bouldin et al., 2010). Loss of Shh signaling from the AER led to an increase in AER length, as determined by measurement of the FGF8 expression domain, and an increased intensity of Shh expression within the ZPA accompanied by formation of an extra postaxial cartilage condensation. Further studies in the chick system showed that implantation of a Shh-soaked bead into the posterior mesoderm adjacent to the ZPA led to a decrease in the length of the AER (Bouldin et al., 2010), although no effect on patterning was observed (Sanz-Ezquerro and Tickle, 2000; Bouldin et al., 2010). These observations have led to proposal of a model for Shh-AER signaling as a mechanism for fine-tuning the level of Shh signaling from the ZPA (Bouldin et al., 2010). In this model, it is predicted that excess Shh from the ZPA would lead to increased Hh signaling in the AER, causing the AER to decrease in length, and resulting in removal of Shh-producing cells from the ZPA by means of induction of apoptosis (Bouldin et al., 2010). In our study, we did not observe reduction in AER length following activation of Shh signaling in the AER of polydactylous Msx2-Shh limbs, nor did we observe reduction in the domain of expression of endogenous Shh in the ZPA, nor down-regulation of Ptch in posterior mesoderm. It is likely, as suggested by Bouldin et al, that the function of Shh-AER signaling in normal limbs to fine-tune limb patterning would result in very subtle changes in AER length and Shh production in the ZPA, because large alterations in either Shh protein or AER length would result in patterning defects. Expression of Shh in the AER of Msx2-Shh limbs in our study leads to induction of Ptch, a sensitive indicator of activation of Shh signaling, throughout the entire AER and in a wide band extending anteriorly across the entire extent of the Msx2-Shh limb mesoderm subjacent to the AER. This indicates that functional Shh protein was produced by the cells of the transgenic AER and released to the underlying mesoderm, activating Shh signaling throughout the distal as well as anterior mesoderm. Thus, activation of Shh signaling throughout the AER and mesoderm of the Msx2-Shh limbs is likely to overwhelm the subtle regulatory loop provided by endogenous Shh-AER signaling. As a consequence, we attribute the phenotypic effects of Shh expression in the AER primarily to the subsequent effects of Shh activation in the mesoderm.

Our analyses of gene expression patterns in limbs from an Msx2-Shh polydactylous line confirmed intense and widespread transgenic Shh expression in the AER, and concomitant ectopic induction of the Shh-responsive gene Ptch in anterior and distal mesoderm, in the high majority of limbs examined. Transgenic Shh was expressed in the AER in a weak or restricted manner in only a small number of limbs, and a similar small number of limbs exhibited normal Ptch expression, suggesting that in these limbs, ectopic AER–Shh expression was not sufficient to induce an ectopic anterior mesodermal signaling center, and providing an explanation for the low frequency of overtly unaffected transgenic limbs we also observed. In addition to ectopic expression of Ptch, the anterior mesodermal signaling center in polydactylous Msx2-Shh limbs was also characterized by ectopic Hoxd11 and Hoxd13 expression, and was accompanied by a widened AER with ectopic anterior expression of Fgf4. The anterior mesodermal domain of Msx2 was suppressed, although Msx2 expression by the AER itself was not. The correlation between absence of Msx2 expression in anterior mesoderm and formation of ectopic preaxial digits is consistent with our studies in the polydactylous limbs of talpid-2 and diplopodia-4 mutant chicks, which lack anterior Msx2 expression (Coelho et al., 1992). Induction of anterior Ptch, Hoxd11, Hoxd13, and Fgf4 expression is typical of many polydactylous phenotypes in which ectopic preaxial digits with various identities are formed. In our study, the least severely affected Msx2-Shh polydactylous limbs possessed extra preaxial digits with anterior identity. This is similar to the extra anterior digits formed in polydactylous diplopodia-4 chicks (Coelho et al., 1992), and in mouse mutants in which Shh signaling in anterior mesoderm is activated through loss of expression of rab23 (Eggenschwiler et al., 2001), thm1 (Tran et al., 2008), tulp3 (Cameron et al., 2009; Norman et al., 2009; Patterson et al., 2009), and Etv4/5 (Zhang et al., 2009), factors which function to repress Shh signaling. Anterior preaxial digits also form following application of the lowest dose of Shh to the anterior margin of the chick limb (Yang et al., 1997), and in mice heterozygous for loss of Gli3 (Hui and Joyner, 1993; Buscher et al., 1998; Chen et al., 2004), suggesting that the preaxial anterior duplications in mildly affected Msx2-Shh limbs result from relatively low-level activation of anterior Shh signaling.

Moderately affected Msx2-Shh limbs display a more severe polysyndactylous phenotype, in which multiple digits are present with similar posterior identity. A strikingly similar polysyndactylous phenotype is observed in the limbs of homozygous Gli3−/− mice (Litingtung et al., 2002; te Welscher et al., 2002; Sheth et al., 2007), suggesting that a higher level of Shh activation causes the polysyndactylous Msx2-Shh phenotype, as opposed to a lower level of Shh activation in mildly affected Msx2-Shh limbs with anterior digit duplication. A type of polydactyly we did not see in our study is the mirror image-type preaxial digit duplications which classically form in chick limb buds following application of a higher dose of Shh protein than that which produces anterior duplications (Yang et al., 1997), or following implantation of an ectopic ZPA to the anterior mesoderm (Lopez-Martinez et al., 1995; Yang et al., 1997; Wada et al., 1999). Mirror-image-type duplications, characterized by digits with posterior identity which form anterior to digit 1, have also been reported in the mouse mutants Rim4, Lst/Alx−/−, and Ssq (Chan et al., 1995; Masuya et al., 1995; Qu et al., 1998; te Welscher et al., 2002; Hill et al., 2003), in which ectopic domains of anterior Shh expression are present. However, the extra preaxial digits of Msx2-Shh limbs are not mirror-image as they form in the absence of digit 1. Lack of mirror-image duplication in Msx2-Shh limbs may reflect the absence of a discretely localized Shh source in anterior mesoderm. Indeed, ectopic cShh was expressed throughout the entire extended AER of the majority of polydactylous Msx2-Shh limbs, including the AER overlying the distal and anterior limb mesoderm, accompanied by induction of Ptch in a wide band subjacent to the AER and extending anteriorly across the entire extent of the distal and anterior mesoderm. A continuous, high level activation of Shh signaling across the posterior, distal, and anterior mesoderm may result in formation of extra digits from distal as well as anterior mesoderm. Consistent with this possibility, implantation of a ZPA into the apex of the chick limb leads to formation of multiple extra distal digits all possessing the same posterior digit identity (Yang et al., 1997), and polysyndactylous limbs strikingly similar to moderately affected polysyndactylous Msx2-Shh limbs are also observed in transgenic mice expressing Shh under the control of the keratin K14 promoter, which is active throughout embryonic ectoderm (Oro et al., 1997). Thus, both the level and the location of the activation of Shh signaling are likely to play a role in generation of distinct polydactylous phenotypes in Msx2-Shh limbs.

A notable difference between these polydactylous phenotypes and our Msx2-Shh mutants is that oligodactyly is not observed following ZPA implantation, application of Shh, or genetic loss of Gli3, Tulp3, or Etv4/5, while in our more severely affected Msx2-Shh mutants, both polydactylous and oligodactylous limbs occur, and can even occur on the same animal. This indicates that activation of Shh signaling in the AER/mesoderm can lead to digit duplication and unexpectedly, also to digit reduction. Typically, digit reduction has been associated with loss of Shh signaling. Oligodactyly is observed in Shh−/− mice (Chiang et al., 1996, 2001; Kraus et al., 2001; te Welscher et al., 2002), in the limbs of chick and mouse mutants lacking Shh expression due to disruption of the Shh-lmbr1 genomic region (Clark et al., 2001; Ros et al., 2003; Maas and Fallon, 2004; Sagai et al., 2005), and in human achieropodia patients in which the lmbr1 gene is mutated (Ianakiev et al., 2001). In our study, however, we observe oligodactyly in the presence of Shh signaling. In this regard, oligodactylous phenotypes have been reported following constitutive activation of Shh signaling in transgenic embryos hypomorphic for Ptch activity (Milenkovic et al., 1999) or lacking limb mesodermal Ptch expression (Butterfield et al., 2009) . In the latter study (Butterfield et al., 2009), loss of limb mesodermal Ptch achieved by Prx1-Cre-mediated deletion results in loss of Ptch from the forelimb earlier than from the hindlimb due to the timing of the Cre transgene (Logan et al., 2002). Intriguingly, the forelimbs of Prx1Cre-Ptchflox embryos were found to be oligodactylous, while hindlimbs were polydactylous (Butterfield et al., 2009), leading the authors to suggest that the timing and resultant level of Hh pathway activation in the limb determines these phenotypic outcomes, such that early and high-level Hh activation leads to oligodactyly, while later and lower-level Hh activation leads to polydactyly (Butterfield et al., 2009). Activation of Shh signaling in the limbs of mice expressing a series of hypomorphic alleles of Twist1, which is thought to function in part to suppress Shh signaling in anterior mesoderm (Krawchuk et al., 2010), supports this suggestion, as progressive reduction of Twist1 activity, accompanied by progressive up-regulation of Shh expression in the anterior mesoderm, led to a progression of limb forelimb phenotypes from polydactyly to oligodactyly (Krawchuk et al., 2010). Analysis of the AP identity of the remaining digit elements in our oligodactylous Msx2-Shh limbs revealed that autopod structures resembling posterior elements were still recognizable, indicating that anterior distal structures were lost. This is consistent with the study of Krawchuk et al. 2010, in which progressive elevation of Shh signaling through increasing gene dosage of Twist1 loss also led to oligodactyly through loss of anterior structures. These observations suggest that, in the present study, the progression of polydactylous to oligodactylous phenotypes in Msx2-Shh mutant limbs is likely the result of progressively increasing levels of Shh activation in the mesoderm. We suggest this is due to differences in timing of expression of the Msx2 transgene, and the subsequent onset and level of Shh produced by the AER which is able to act on the underlying subridge mesoderm to produce constitutive mesodermal activation of Shh signaling. Because the Msx2 promoter drives expression of Shh to the hindlimb when it is at an earlier stage of development than the forelimb (Sumoy et al., 1995), hindlimbs are exposed to an earlier onset and hence presumably a cumulatively higher level of Shh activation than forelimbs, consistent with our observation that limb defects are more severe in Msx2-Shh hindlimbs than forelimbs. In addition, because the Shh pathway in Msx2-Shh limbs is constitutively activated by Msx2-promoter-driven Shh expression, it cannot be controlled by endogenous regulators such as Ptch, Tulp3, or Etv4/5, resulting in continuous and presumably high-level activation of Shh signaling consistent with our ability to generate severe oligodactylous phenotypes not typically seen following individual loss of Shh pathway inhibitors. Together, our observations underscore the importance of the control of the timing and level of activation of Shh signaling for proper limb AP patterning (Harfe et al., 2004; Scherz et al., 2007; Bastida and Ros, 2008; Towers et al., 2008; Zhu et al., 2008; Yang, 2009).

We also observed a spectrum of defects in proximal patterning in our Msx2-Shh limbs. The least severe proximal defects occurred in conjunction with polydactyly and included a shortened hindlimb zeugopod and missing anterior structures (i.e., tibia). More severe proximal defects occurred in conjunction with oligodactyly, and included in forelimbs, shortened and fused zeugopod elements and missing stylopod, and in hindlimbs complete loss of zeugopod and stylopod elements, resulting in a phocomelic phenotype. Mild proximal patterning defects have also been observed in several of the polydactylous mouse mutants in which Shh pathway signaling is inappropriately activated in the limb including Rim4−/− and Hx (Masuya et al., 1995; Blanc et al., 2002) in which the hindlimb zeugopod is shortened; and Alx4−/−/lst (Qu et al., 1998), Xt/Gli3−/− (Masuya et al., 1995; Litingtung et al., 2002) and Ssq (Sharpe et al., 1999; Hill et al., 2003), in which the hindlimb zeugopod is shortened and part or all of the tibia is missing. Similar relatively mild defects consisting of shortened zeugopod and loss of anterior structures (radius/tibia) are also observed in the limbs of Twist1 mutant mice with low-dose gene inactivation (Krawchuk et al., 2010). Intriguingly, the severe proximal defects and phocomelia observed in Msx2-Shh forelimbs are remarkably similar to the severe proximal defects which occur in Twist1 mutant forelimbs (Krawchuk et al., 2010), in which Prx1Cre-mediated loss of Twist1 occurs at a very early stage (Logan et al., 2002), and in which strong and widespread activation of mesodermal Shh signaling results in reduced FGF signaling to the AER and a subsequent reduction in the length of the AER as determined by the extent of Fgf8 expression (Krawchuk et al., 2010). The phenotypic similarity between Twist1 mutant forelimbs and our severely affected Msx2-Shh limbs suggests that Shh activation in severely affected Msx2-Shh limb mesoderm is occurring at a very high level, and may be similarly impairing maintenance of the AER, resulting in shortening of the AER and causing proximal defects and limb truncation. Although due to poor fertility we were unable to obtain oligodactylous Msx2-Shh limbs at embryonic stages in which to examine AER length, the distal tips of newborn severely oligodactylous Msx2-Shh fore- and hindlimbs are clearly reduced in width, consistent with prior shortening of the AER.

An alternate possibility is that the presumably high-level activation of Shh signaling in the limb mesoderm of the most severely affected Msx2-Shh limbs causes a loss of progenitor cells in the zeugopod and stylopod such that insufficient cells are available to undergo the condensation process necessary for chondrogenic differentiation. This mechanism has been suggested to underlie the formation of phocomelic limbs resulting from teratogenic effects of thalidomide use in humans (Newman, 1986) and X-irradiation of the developing limb in chicks (Galloway et al., 2009), which like the most severely affected Msx2-Shh limbs, possess autopods but lack zeugopod and/or stylopod elements (Galloway et al., 2009). According to this mechanism, elimination of chondrogenic precursors during a time window when formation of the proximal condensations is occurring, but distal differentiation has not yet begun, leads to subsequent loss of proximal but not distal elements (Galloway et al., 2009). Loss of proximal chondrogenic progenitors could occur due to increased mesodermal cell death in the proximal region by means of loss of AER–FGF signaling, as has been suggested for the proximal defects observed in limbs lacking AER expression of both FGF4 and FGF8 (Mariani et al., 2008). Although we were unable to examine cell death in severely affected Msx2-Shh limbs, because massive cell death of the proximal region requires combined loss of FGF4 and FGF8, and because we found that both FGF4 and FGF8 continue to be expressed by the AER of at least the less-severely affected Msx2-Shh limbs, despite simultaneous AER expression of Shh, it seems unlikely that complete loss of both FGF4 and FGF8 would occur even in the most severely affected Msx2-Shh limbs. Intriguingly, a recent study has demonstrated that limb mesenchymal cells obtained from Prx1Cre-Ptchflox embryos and maintained in micromass culture form apparently normal precartilage mesenchymal condensations, but that subsequent chondrogenic differentiation is impaired (Bruce et al., 2010), indicating that inappropriate activation of Hh signaling in early limb mesenchyme disrupts the chondrogenic differentiation process. This raises the possibility that in severely affected Msx2-Shh limbs, inappropriate and high level activation of Shh signaling in limb bud mesoderm at early stages of limb development, when chondrogenic differentiation of the proximal structures is occurring, may result in impaired chondrogenic differentiation and subsequent loss of proximal skeletal elements. As our studies indicate that Msx2 promoter-driven lacZ expression is reduced in the limb distal ectoderm overlying the regressing interdigital mesoderm around E13.5–E14.5, and is only retained by the distal ectoderm overlying the digit tips, a reduced level of Shh activation during the time that chondrogenic differentiation of the autopod elements is occurring could permit formation of distal structures, albeit abnormally patterned ones, in severely affected Msx2-Shh limbs. Recent studies demonstrating that distinct thresholds of Shh activation define digit number (Hill et al., 2009) are consistent with the possibility that subtle temporal, spatial and quantitative changes in Shh activation in the limb can be translated into dramatic effects on the AP or PD patterning of the limb skeletal elements (Harfe et al., 2004; Scherz et al., 2007; Bastida and Ros, 2008; Towers et al., 2008; Zhu et al., 2008; Yang, 2009).

According to the model of Bouldin et al. (2010), a low and localized activation of Shh signaling in the AER is predicted to lead to down-regulation of Shh expression by the ZPA, followed by loss of mesodermal progenitors otherwise capable of forming skeletal tissue. Thus, it could be reasoned that oligodactyly in Msx2-Shh limbs could result from low and localized ectopic AER cShh expression insufficient to activate Shh signaling ectopically in the underlying mesoderm, such that oligodactyly would result from a reduced rather than increased activation of Shh signaling in the limb mesoderm. We believe this is an unlikely possibility. Although we do observe a low frequency of limbs with weak or restricted ectopic cShh expression, we never observed down-regulation of endogenous Shh or of endogenous Ptch. This indicates that endogenous Shh signaling is intact in these limbs and is not being down-regulated. Moreover, if mesodermal progenitors in the ZPA were being removed, because these cells have been found to contribute primarily to posterior digit structures (Harfe et al., 2004), it would be predicted that posterior digit elements would be lost and anterior digits unaffected in oligodactylous Msx2-Shh limbs, which is the opposite of what we observe in our study, which is that anterior structures are lost while posterior structures remain. In addition, we found that in our study oligodactyly occurred with very high frequency in conjunction with polydactyly, especially severe polydactyly, but nearly never with unaffected limbs. Oligodactyly in Msx2-Shh limbs is also accompanied by severe proximal patterning defects resulting in a phocomelic phenotype. Thus, oligodactyly in our study is a component of the most severe phenotype among the spectrum of phenotypes in Msx2-Shh limbs. It should also be pointed out that phocomelia is not a component of the oligodactylous phenotypes of limbs in which Shh signaling is lost, such as Shh−/− mice and chick ozd mutants (Kraus et al., 2001; Ros et al., 2003). Together, these observations argue in favor of our hypothesis that the severe oligodactylous and phocomelic phenotype in Msx2-Shh limbs is the result of very high-level activation of Shh signaling in the underlying limb mesoderm, and not due to reduction in mesodermal Shh signaling.

Surprisingly, dorsoventral patterning was also disrupted in the limbs of the most severely affected Msx2-Shh animals, which displayed ventral footpads present on the dorsal limb surface as revealed by dermatoglyphic analysis. Ventral footpads on the dorsal limb surface are also a feature of Wnt7a−/− mice (Cygan et al., 1997; Parr et al., 1998). The presence of ventral footpads on the dorsal surface of Msx2-Shh limbs suggests that constitutive high level activation of Shh in limb AER and mesoderm leads to partial ventralization of the limb. To our knowledge, this has not been previously reported. Although dorsal ectoderm is needed to maintain Shh expression in the limb (Parr et al., 1998), the reverse has not been shown and indeed, the dorsal markers Wnt7a and lmxb are expressed normally in ozd limbs and in Shh−/− limbs (Chiang et al., 2001; Kraus et al., 2001; Ros et al., 2003). However, Shh signaling has long been recognized as having a critical role in DV patterning of the neural tube where it is required to promote ventral identities (Jessell, 2000; Wijgerde et al., 2002; Ulloa and Briscoe, 2007; Fogel et al., 2008). Inappropriate activation of Hh signaling in the neural tube and/or hindbrain following loss of Hh pathway inhibitors such as Ptch (Goodrich et al., 1997; Milenkovic et al., 1999), Gli3 (Persson et al., 2002; Lebel et al., 2007), rab23 (Eggenschwiler et al., 2001), Thm1 (Tran et al., 2008), or Tulp3 (Cameron et al., 2009; Norman et al., 2009; Patterson et al., 2009) leads to expansion of ventral cells at the expense of dorsal ones, and causes severe CNS defects typified by open brains or neural tubes. This may relate to the defects in hindbrain development we commonly observe in Msx2-Shh embryos, as we have found that the Msx2 promoter drives lacZ expression not only in the limb AER, but also in a high percentage of embryos, in the hindbrain ectoderm (Sumoy et al., 1995). These observations are consistent with a requirement for control of Shh signaling for normal CNS development, and suggest the novel possibility that regulation of Shh activity in the limb is also required for proper DV patterning.

EXPERIMENTAL PROCEDURES

Msx2-LacZ and Msx2-Shh Transgene Construction and Generation of Transgenic Mice

A construct was made in which an 800 bp BglII/SmaI region containing the AER enhancer and promoter of the chicken Msx2 gene (Sumoy et al., 1995) was replaced with a 5.5 kb KpnI/SmaI fragment extending 5′ from the transcription start site to generate the Msx2-LacZ construct used in this study (Fig. 1). The Msx2-Shh transgene was made by digesting the Msx2-LacZ construct with XbaI and Bpu11021I and inserting a full coding 1.4 kb XbaI/NotI chicken Shh cDNA fragment from pHh-2 (Riddle et al., 1993) in place of the LacZ gene. Use of chicken rather than mouse Shh coding sequences enabled subsequent distinction between transgenic and endogenous Shh expression (Fig. 5). Transgenic mice were generated in FVB/N embryos as described previously (Sumoy et al., 1995). Transgenic mice and embryos were initially identified by the presence of limb abnormalities and/or hindbrain defects and subsequently confirmed to be transgenics by DNA analysis. The presence of hindbrain defects likely contributed to a high rate of neonatal death of the founders. Surviving founders had poor fertility. Of the five transgenic lines established, only one line (polydactylous line 13) produced sufficient embryos for further phenotypic analysis of the progression of limb digit formation and expression of patterning genes.

Phenotypic Analysis

For analysis of limb skeletal elements, limbs were stained whole-mount with Alcian blue and Alizarin red to visualize cartilage and bone, respectively (McLeod, 1980). Tissue section in situ hybridization was performed as described previously (Coelho et al., 1991). Probes used for in situ hybridization were as follows: chicken Shh cDNA clone pHh-2 (Riddle et al., 1993), mouse Shh cDNA (Echelard et al., 1993), mouse Ptc cDNA (Goodrich et al., 1996), mouse Fgf4 cDNA clone K1 (Niswander and Martin, 1992), mouse Msx2 cDNA clone pSP72/MHox-8 (MacKenzie et al., 1992), and mouse Hoxd11 cDNA pGem1-AccI-BamHI (Izpisua-Belmonte et al., 1992). For dermatoglyphics on the volar skin, limbs were fixed in 10% formalin for 3 or more days at room temperature and stored until they were further processed as described by (Tsugane and Yasuda, 1995).

Ancillary