Shh signaling in limb bud ectoderm: Potential role in teratogen-induced postaxial ectrodactyly

Abstract

A variety of teratogens induce the loss of postaxial forelimb structures when administered during mid-gestation to the mouse. Previous studies demonstrated that teratogen exposure is associated with a reduction in zone of polarizing activity (ZPA) -related polarizing activity without a noticeable loss of Shh expression. Herein, we quantitatively confirm that expression of Shh, Ptch1, and Gli3 are unaltered by teratogen exposure and demonstrate that sonic hedgehog (Shh) translation is unaffected. Examination of the polarizing response of host chick wings to teratogen-exposed ZPA tissue revealed an induced growth response and ectopic induction of Fgf4, Bmp2, Ptch1, and Gli1 expression similar to control ZPA tissue. Control ZPA tissue altered the fate of cells destined to die in the anterior necrotic zone, whereas cell death ensued in hosts receiving teratogen-exposed grafts. Immunohistochemical studies localized Shh protein in the mouse limb to the posterior mesoderm and overlying ectoderm. We postulate that teratogen exposure alters the ability of Shh to signal to the ectoderm and present microarray and reverse transcriptase-polymerase chain reaction data, indicating that Shh signaling could occur in the limb bud ectoderm. Developmental Dynamics 233:313–325, 2005. © 2005 Wiley-Liss, Inc.

INTRODUCTION

The developing limb bud postaxial mesoderm of the chick (Saunders and Gasseling, 1968), mouse (Tickle et al., 1976; Wanek and Bryant, 1991), and other amniotes (Fallon and Crosby, 1977) includes the zone of polarizing activity (ZPA). By definition, transplantation of a tissue that possesses polarizing activity into the anterior margin of a host chick wing bud induces mirror-image digit duplications. The cellular activities required to be induced in the host tissue by a ZPA graft include growth (cell proliferation) and pattern formation. The question of whether these two cellular functions are controlled by a single or multiple signals was discussed by Cooke and Summerbell (1980). At that time, the evidence suggested that multiple signals was the more likely choice. Subsequently, it has been shown that sonic hedgehog (Shh) is a primary component of polarizing activity. This idea is supported by the following observations: (1) the domain of Shh expression in the limb bud is spatially and temporally coincident with that of the ZPA in both the chick and the mouse (Riddle et al., 1993; Chang et al., 1994); (2) other tissues that express Shh also possess polarizing activity (Wagner et al., 1990; Izpisua-Belmonte et al., 1992; Chang et al., 1994; Koyama et al., 1996; Selleck et al., 1996); (3) in the presence of the apical ectodermal ridge (AER), ectopic introduction of Shh into the anterior margin of a chick wing bud induces duplications typical of ZPA grafts (Riddle et al., 1993; Lopez-Martinez et al., 1995; Yang et al., 1998); (4) the AER is required to maintain polarizing activity and Shh expression (Niswander et al., 1993, 1994); (5) ZPA grafts and ectopic Shh induce ectopic anterior domains of Fgf4, Hoxd-13, Ptch1, and Bmp2 (Izpisúa-Belmonte et al., 1991; Francis et al., 1994; Niswander et al., 1994; Marigo et al., 1996b; Yang et al., 1997); (6) Shh has been shown to possess proliferative and cell fate activity in other developing organ systems (Roelink et al., 1994; Bellusci et al., 1997; Roberts et al., 1998; Ahlgren and Bronner-Fraser, 1999; Haraguchi et al., 2001).

Despite this impressive evidence, our previous observations made us question whether there is a direct relationship between Shh and polarizing activity because exposure to select teratogens could induce the loss of posterior digits, abolish polarizing activity but not noticeably affect Shh expression in the posterior limb bud mesenchyme (Bell et al., 1999; Scott et al., 2005). We further showed that the expression of Patched (Ptch1), the hedgehog receptor whose expression is exquisitely sensitive to Shh activity, was unaltered by teratogen exposure.

The present studies examine Shh status in the teratogen-exposed mouse limb bud and the cellular and molecular changes in the host chick limb after grafting teratogen-exposed or control mouse embryo ZPA tissue. Our findings suggest that teratogen exposure disrupts Shh signaling by a mechanism independent of effects on Shh transcription/translation and that physiological doses of Shh required to induce ectopic domains of downstream target genes are insufficient to alter the developmental fate of cells within the anterior necrotic zone (ANZ) of the host chick wing. We also suggest that teratogen exposure alters the ability of Shh to directly signal to the posterior limb ectoderm/AER and that it is the alteration of this event not the loss of polarizing activity that leads to the loss of posterior structures.

RESULTS

Our previous studies indicated that stage 3 and 4 mouse limb ZPA tissue exposed to acetazolamide had dramatically diminished polarizing activity in the presence of a Shh expression domain that was similar to those observed in stage-matched untreated forelimb buds (Bell et al., 1999). To further define the cause of reduced polarizing activity induced by teratogen exposure, four lines of investigation were pursued: (1) the response of the chick host anterior marginal tissue to the acetazolamide-exposed graft was evaluated, (2) the levels of Shh and downstream effector expression were quantitated in treated and untreated mouse limb buds, (3) Shh protein production by the treated murine limb ZPA tissue was evaluated, and (4) functionality of the murine Shh protein was evaluated using another activity assay.

Response of the Chick Anterior Mesoderm to a ZPA Graft

Previous studies revealed that ZPA tissue (Smith, 1980), beads embedded with mouse Shh (Yang et al., 1997), or retinoic acid (Eichele et al., 1985) usually failed to induce digit duplications if in contact with the wing anterior mesenchyme for less than ∼15 hr. Therefore, the failure of teratogen-exposed ZPA tissue to induce duplications could be explained by a premature loss of Shh expression by the graft. Whole-mount in situ hybridization (WMISH) studies indicated that Shh expressing tissue isolated from stage 3 acetazolamide-exposed limb buds continued to express Shh for >24 hr after grafting (Fig. 1A,B; Table 1; Bell et al., 1999). This length of exposure to either ZPA tissue or beads harboring mouse Shh is normally sufficient to induce full mirror-image duplications, yet not even an additional digit 2 was routinely induced by teratogen-exposed ZPA tissue.

Figure 1. Ectopic gene expression after zone of polarizing activity (ZPA) grafting to stage 19/20 host chick wing buds. A–F: Whole-mount in situ hybridization 24 hr after transplantation of either untreated (A,C,E) or acetazolamide-exposed (B,D,F) ZPA tissue from stage 3 mouse limb buds. A,B: Arrows indicate Shh expression in mouse ZPA grafts. A–D: By 24 hr, ectopic anterior apical ectodermal ridge (AER) expression of Fgf4 has been induced overlying the grafts (arrowheads, A,B) and Bmp2 is induced in the mesoderm distal to the graft (arrows, C,D). Asterisk in C and D indicate graft location. E,F: Arrows indicate up-regulated Ptch1 expression around the graft. G–J: Method of limb measurement 30 hr after ZPA grafting. Ventral aspect of all limbs. G,I: Contralateral ungrafted left limbs (G,I). Right limbs 30 hr after receiving either a control (F) or an acetazolamide-exposed (H) ZPA graft. Lines indicate the position of the base of the limb, proximal/distal length, and anterior/posterior length. At this time point both control and acetazolamide-exposed ZPA tissue induced expansion of the anterior limb margin when compared with the ungrafted left wing of the same host. A (anterior margin), P (posterior margin).

Table 1. Gene Expression in and Around ZPA Grafts Made to the Anterior Wing Margin

Graft origin	Shh*	Fgf4 (+)	Bmp 2 (+)	Gli1 (+)	Ptch1 (+)	Gre (+)	Bmp4 (−)
* For Shh, expression is within the graft. The expression of all other genes is in the anterior mesoderm and AER. Plus signs indicate up-regulated expression. Minus signs indicate that the normal domain was down-regulated. Values in parentheses indicate number of grafts made. ZPA, zone of polarizing activity.
Control ZPA
24 hr	14 (16)	4 (7)	3 (4)	6 (6)	11 (11)	2 (2)
30 hr	10 (12)	3 (4)			5 (6)
36 hr	4 (10)						3 (4)
Acetazolamide-exposed ZPA
24 hr	11 (19)	5 (6)	4 (6)	5 (7)	4 (7)	1 (4)
30 hr	5 (12)	3 (5)			2 (4)
36 hr	7 (19)						0 (4)

In response to ZPA grafts or exogenous Shh, ectopic anterior expression domains of Fgf4, Ptch1, Bmp2, and Hoxd cluster genes are up-regulated suggesting that these genes are indicators of effective Shh signaling in the limb bud (Izpisua-Belmonte et al., 1992; Riddle et al., 1993; Francis et al., 1994; Laufer et al., 1994; Lopez-Martinez et al., 1995; Marigo et al., 1996b; Yang et al., 1997). Like vehicle control ZPA tissue, acetazolamide-exposed ZPA tissue induced ectopic anterior AER expression of Fgf4 within 24 hr (Fig. 1A,B; Table 1). Within the mesoderm, ectopic domains of Bmp2, Gli1, and Ptch1 were also observed in the tissue between the graft and the AER in response to both control and acetazolamide-exposed ZPA tissue (Fig. 1C–F, data not shown). Ptch1 expression was up-regulated in response to all control ZPA grafts, but the intensity and area of the up-regulated anterior expression domain varied considerably between individual hosts, including being positioned immediately around the graft (Fig. 1E) to a broad domain expanding distally along the anterior limb margin (data not shown). A slightly lower percentage of host chick wings exhibited Ptch1 expression after receiving a ZPA graft from a teratogen-exposed ZPA (Table 1). However, in those hosts that did exhibit ectopic Ptch1 expression, the up-regulated domain of expression was usually restricted to the region immediately around and slightly distal to the graft (Fig. 1F).

Gli1 is thought to be a positive effector of Shh signaling. In response to a control ZPA graft, there is a symmetrical episode of Gli1 induction around the graft that varies in intensity among individuals. Gli1 induction in response to a teratogen-exposed ZPA graft was detectable, but the intensity and expanse of the induced area of expression was often less than that observed in response to a control graft (Table 1, data not shown).

Cooke and Summerbell (1981) previously observed that within ∼8 hr of grafting ZPA tissue the proliferation rate of the anterior mesenchyme increases, resulting in a widening of the anterior margin morphologically visible by ∼24 hr. During our studies to monitor gene expression changes in the chick wing after grafting, it became evident that widening of the wing was occurring in response to teratogen-exposed ZPAs, even though no subsequent limb duplications were found (Fig. 1H). This growth is centered around the grafted ZPA tissue and consequently widens the anterior–posterior axis of the limb. We made measurements of the limb width 30 hr after grafting using the method of polar coordinates previously described by Smith and Wolpert (1981) and depicted in Figure 1G–J. Photographs of wing buds processed for WMISH 30 hr after grafting were used to measure the proximodistal length and anterior posterior width of the grafted right wing bud and contralateral control left wing bud of each chick embryo. As a control for a wound-induced growth response, anterior mesenchyme from untreated stage 3 mouse limb buds was also grafted. As presented in Table 2, the measurements indicate that the anterior tissue fails to induce a growth response in the host chick wing bud because the width of grafted right wings and unmanipulated left wings is the same. Upon grafting ZPA tissue isolated from stage 3 forelimb buds from a control or teratogen-exposed mouse embryo, the width of right limbs was found to be increased in comparison to left limbs (Fig. 1G vs. H and I vs. J). However, it should be noted that the increased measured widths were somewhat less in response to a teratogen-exposed ZPA graft suggesting that the growth-inducing properties of the ZPA tissue from teratogen-exposed ZPA tissue may be slightly less than that of control ZPA tissue. By 42 hr, right limbs that received a control graft are dramatically enlarged anteriorly (Fig. 2C,F). In contrast, usually only a slight difference in morphology is observed between the ungrafted left limb and a right limb, which received a treated ZPA graft (Fig. 2A vs. B, D vs. E).

Table 2. Dimensions (mm) of Chick Limbs 30 hr After Grafting Mouse Mesoderm to a Chick Host Wing*

	Right limb	Left limb
* A/P, anterior–posterior; ZPA, zone of polarizing activity; PD, posterior–dorsal.
	Width (A/P axis)
Control ZPA (n = 12)	1.31 ± .14	1.09 ± .10
Acetazolamide ZPA (n = 13)	1.24 ± .10	1.09 ± .09
Anterior mouse mesoderm (n = 5)	1.05 ± .04	1.00 ± .04
	Length (P/D axis)
Control ZPA	1.38 ± .14	1.34 ± .11
Acetazolamide ZPA	1.24 ± .13	1.21 ± .12
Anterior mouse mesoderm	1.26 ± .06	1.30 ± .10

Figure 2. Changes in anterior margin cell death, Bmp4, and Gre in response to zone of polarizing activity (ZPA) grafts. A,D,G: Ungrafted left wings. B,C,E,F,H,I: Right wings that received either control (C,F,I) or acetazolamide-exposed ZPA grafts (B,E,H). A–C: Ventral views of chick wings stained with Nile blue sulfate 42 hr after ZPA tissue was grafted into the right wing of stage 19/20 hosts. D–G:Bmp4 expression in limbs 36 hr after grafting. H,I:Gre expression 24 hr after grafting. Note the expanded anterior margins in C and F; the absence of an ANZ in C; and diminished Bmp4 expression in F. An ANZ is present in teratogen-exposed ZPA grafted limbs that is elongated when compared with the ANZ of the left limb. In A–F, the anterior margin is up. Arrowheads in A–C indicate boundaries of the ANZ. Arrowheads in D–F indicate position of Bmp4 expression domain. Arrows in H and I indicate up-regulated Gre expression in the distal mesenchyme.

As reviewed by Hinchliffe and Johnson (1980), there are normally numerous episodes of cell death that occur during the development of the chick limb. One of these episodes occurs in the anterior marginal limb mesenchyme, the ANZ. Cell death in this zone begins at stage 23 in the proximal wing mesenchyme, wanes during stage 24, and then reappears in greater extent distally during stage 25/26. Using the uptake of Nile blue sulfate as an indicator of cell death, we examined the effects of control and teratogen-exposed ZPA grafts on the ANZ at 30, 36, and 42 hr after grafting. Thirty hours after grafting, embryos possess stage 22–24 wing buds and small regions of Nile blue staining were usually observed in the unmanipulated left wing bud of host chick embryos. In response to either a control mouse ZPA graft or acetazolamide-exposed ZPA graft, ∼70% of the grafted wings lacked a detectable ANZ (Table 3). An ANZ was consistently observed in all host chick left wings at 36 and 42 hr after grafting (Fig. 2A; Table 3). In response to control ZPA tissue grafts, an ANZ was rarely observed in host right wings at 36 hr. Nile blue uptake in host right wings receiving teratogen-exposed ZPA tissue was more varied at 36 hr. An ANZ was absent in 37% of the hosts. In the remaining limbs, an ANZ was detectable, however, when the right limb was compared with the host left limb, it was smaller in 20% of the hosts, elongated in 17% of the hosts, or comparable in size to the left, 27%. At 42 hr after grafting, control ZPA tissue cell death was not detected at all in half of the grafts; however, a small domain of cell death located proximal to the graft site was observed in six wings. Forty-two hours after receiving a teratogen-exposed ZPA graft, an ANZ was detected in most limbs and was elongated along the proximal– distal axis in half of the limbs examined (Table 3; Fig. 2A vs. B). To control for cell death-induced changes attributable to the surgical manipulation, we also grafted mouse anterior limb mesenchyme into host chicks and examined Nile blue uptake at 36 hr in 12 grafted limbs. Normal-sized ANZs were observed in 11 of 12 limbs, whereas 1 limb possessed a smaller ANZ than its contralateral control limb. Combined, these findings indicate that signals derived from a control ZPA graft alter the developmental fate of ANZ cells and that the commitment to an altered cell fate is not maintained by tissue responding to a teratogen-exposed ZPA graft, although these grafts do slightly delay the onset of ANZ cell death.

Table 3. Cell Death Observed in Host Right Wings Receiving a Graft Versus the Left Winga

Time after grafting (hr)	ANZ appearance	Control ZPA		Acetazolamide-exposed ZPA		Anterior mesoderm
		Left	Right	Left	Right	Left	Right
a ANZ, anterior necrotic zone; ZPA, zone of polarizing activity. b Small domain proximal to the graft.
30	None		8	1	8
	Normal	11	3	12	4
36	None		17		11		1
	Small		1b		6
	Elongated				5
	Normal	18		30	8	12	11
42	None		6		1
	Small		6b		4
	Elongated				8
	Normal	12		19	6

Episodes of programmed cell death in the vertebrate limb bud and other organs have been associated with Bmp4 expression (Yokouchi et al., 1996; Zou and Niswander, 1996; Macias et al., 1997; Ganan et al., 1998; Pizette and Niswander, 1999). Moreover embryos heterozygous for a Bmp4 null mutation exhibit preaxial polydactyly. Furthermore, embryos heterozygous for Bmp4 and Gli3 exhibit preaxial polydactyly as well as an absence of cell death in the preaxial subridge mesoderm (Dunn et al., 1997). Thus, we analyzed Bmp4 expression at multiple postgrafting intervals in host chick wings. As previously described, Bmp4 expression in the unoperated left wing was heavy in the AER and anterior marginal mesenchyme (Yokouchi et al., 1996). We did observe two expression domains in the anterior mesenchyme, one dorsal and one ventral (Fig. 2G), the dorsal domain being expressed earlier than the ventral domain. Bmp4 expression was profoundly affected by a ZPA graft from control tissue and these changes were most distinct 36–42 hr after grafting (Fig. 2D vs. F). Bmp4 expression was limited to the anterior mesenchyme on either side of the ectopic outgrowth and only the dorsal arm of staining was present. In contrast, Bmp4 expression in the chick wing 36–42 hr after a ZPA graft from an acetazolamide-exposed mouse limb bud did not alter Bmp4 expression from that of the contralateral unoperated limb (Fig. 2D vs. E).

Bmp signaling is negatively controlled by a variety of extracellular and cellular antagonists (see Balemans and Van Hul, 2002, for review). In the vertebrate limb bud, a major influence on Bmp signaling is provided by Gremlin (Gre), whose expression is regulated by Shh signaling (Capdevilla et al., 1999; Merino et al., 1999; Zuniga et al., 1999; Khokha et al., 2003; Michos et al., 2004; Scherz et al., 2004). Thus, we examined Gre expression in the chick wing after ZPA grafts from vehicle or acetazolamide-exposed mouse embryos. In the chick wing with a control graft, Gre expression was up-regulated in tissue adjacent to the graft but especially distal to the graft where the digit duplication will occur (Fig. 2I). In contrast, chick wings grafted with a ZPA from an acetazolamide-exposed mouse embryo revealed little or no up-regulation in anterior Gre expression (Fig. 2H). Thus, in response to a control ZPA graft, Bmp signaling would be diminished in the distal mesenchyme of the chick wing through repression of Bmp4 expression and up-regulation of Gre expression.

The product of the Msx2 homeobox gene has been associated with cell death in the vertebrate embryo through a Bmp4-mediated pathway (Graham et al., 1994). We examined Msx2 expression 24, 36, and 42 hr after grafting and found that a ZPA graft from a control mouse embryo led to a significant reduction of staining in the anterior mesoderm. Grafting of a ZPA from a teratogen-exposed mouse embryo did not alter Msx2 expression corresponding to normal Bmp4 expression and detection of an ANZ (data not shown).

Effect of Teratogen Exposure on Murine ZPA Shh/Shh Content and Signaling Activity

Because whole-mount in situ hybridization assays are qualitative at best, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on E10.5 limb bud total RNA isolated from embryos exposed to either the vehicle or acetazolamide on E9.5. PCR was performed on two independent treated and vehicle biological samples using gene-specific primers for either Shh, Ptch1, or Gli3. Gene-specific PCR reactions were normalized to levels of α-tubulin present within the same samples. As presented in Figure 3A, real-time RT-PCR indicates there is little or no reduction in the level of Shh, Ptch1, or Gli3 induced by exposure to acetazolamide.

Figure 3. Sonic hedgehog (Shh) signaling components in the mouse limb. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) quantitation of Shh and its signaling components in the embryonic day 10.5 limb bud. B: Western blot analysis of Shh in acetazolamide-exposed and control posterior halves of limb buds. C: Shh signaling assay using Shh-LIGHT2 cells.

Although Shh expression appears normal by RT-PCR, teratogen exposure could prohibit normal Shh translation. To determine whether similar levels of Shh protein are produced by acetazolamide- and vehicle-exposed E10.5 limb buds, we performed Western blot analysis using limb bud extracts from the posterior half of the limb. Blots were probed with an anti-Shh antibody and reprobed with an antibody to β-tubulin to control for the levels of protein loaded in each lane. Figure 3B indicates that similar levels of Shh protein are made by the teratogen-exposed limb bud.

Our previous studies indicated that polarizing activity, one assay of Shh signaling ability, was dramatically diminished by teratogen exposure. As an additional assay of Shh activity, cell lysates from E10.5 vehicle and acetazolamide-exposed limb buds were applied to Shh LIGHT2-cells, a stable cell line containing both firefly luciferase under control of a Gli1-inducible promoter and Renilla luciferase under control of the thymidine kinase promoter (Taipale et al., 2000). In three independent experiments, acetazolamide-exposed limb bud lysates had only 22% of the luciferase activity induced by control lysates (Fig. 3C). These findings suggest that, although Shh is transcribed and translated normally in teratogen-exposed limbs, its biological activity is diminished.

Shh Signaling to the Ectoderm

Traditionally, Shh signaling is thought to occur within the limb bud mesenchyme. One effect of teratogen-exposure is not only a loss of Shh signaling but also a loss of the posterior AER (Bell et al., 1999). Shh is postulated to maintain the AER by induction of Gre (Merino et al., 1999; Zuniga et al., 1999; Khokha et al., 2003; Michos et al., 2004). WMISH assays indicated that Gre expression is normal in teratogen-exposed mouse limb buds (data not shown). This observation in combination with the observed reduction in Shh signaling activity and loss of the posterior AER suggested Shh may signal directly to the ectoderm. To evaluate components of the Shh signaling pathway present within the emerging limb ectoderm during this window of development, we have performed two microarray experiments on ectodermal hulls. Presented herein is an experiment in which ectodermal hulls were isolated from stage 1.5 and stage 4 limb buds and the data examined on the Affymetrix MOE 430 v2.0 chip containing most of the mouse transcriptome. In a previous experiment, ectodermal hulls were isolated from both vehicle control and acetazolamide-exposed embryos 3 and 12 hr after exposure but only analyzed on the Affymetrix U74A v2.0 chip. For some genes, multiple probe sets are present on the MOE 430 v2.0 chip, with conflicting absent versus present calls. The discrepancy among probe sets for the same gene could be attributable to inefficient reverse transcription of more 5′ regions of the genes. Alternatively, in our experience, some probe sets poorly detect messages known to be present within a sample. We considered a gene to be expressed if one or more of the probe sets considered the gene to be present. If a gene was considered present by a target probe set, the expression levels detected were never borderline but were detected at a level comparable to known limb ectoderm genes, including Fgf8, En-1, and Wnt7a. Using these criteria, multiple components of the Shh signaling pathway are expressed by the limb ectoderm, including Smoothened (Smo), Ptch1, Ptch2, Gli1, Gli2, and Gli3. Notably, as Shh expression comes on in the limb mesenchyme between stage 1.5 and stage 4, there is a corresponding up-regulation in Ptch1, Ptch2, and Gli1 expression in the overlying ectoderm (Table 4); whereas, expression of Shh was not detectable. Nor was expression of other mesenchymal markers, including Hoxd11 and Hoxd12, suggesting that the detected expression of Ptch1, Ptch2, Smo, and Gli family members is due to expression by the limb ectoderm and not attributable to mesenchymal contamination of the sample. Furthermore, the expression of Smo and Ptch1 were also detected in our six vehicle control and six acetazolamide-exposed ectoderm samples applied to the U74A chip—however, teratogen exposure did not alter the levels of gene expression (data not shown). Because the presence of Ptch1, Smo, and Gli family members in the early limb ectoderm was surprising, we performed real time RT-PCR on total RNA isolated from ectodermal hulls containing the dorsal/ventral and AER compartments of the limb ectoderm. Gene-specific primers spanning one or more introns within the coding region were used to detect the presence of Ptch1 and Gli3 in the ectoderm samples. Gene expression levels were normalized to α-tubulin (Fig. 4A). The values obtained confirmed the microarray data indicating that Gli3 is present at both developmental stages and that the level of Ptch1 expression is increased approximately twofold in the stage 4 limb ectoderm compared with the stage 1 limb ectoderm sample. Agarose gel analysis of the amplicons revealed that the products generated were of the correct size (Fig. 4B). Combined, these data indicate that Ptch1 and Gli3 are expressed by the limb ectoderm.

Table 4. Expression of Genes in Emerging Limb Ectoderm

Gene	Affymetrix probe ID	Stage 1.5		Stage 4
		Average gene expression level (average P value)	Call	Average gene expression level (average P value)	Call
a The absolute call (Call) was considered Absent (A) or Present (P) in two of the three samples. c The call was considered marginal in one, absent in another, and present in the third sample. b Position of the unique probe set relative to the other probe sets along the target cDNA.
Shh	1427571_at	105 (0.2283)	A	101 (0.2283)	A
	1436869_at	165 (0.3907)	A	540 (0.0825)	Aa
Smo	1442215_at 5′b	55 (0.3369)	A	27 (0.5753)	A
	1427048_at Middle	7,533 (0.0004)	P	7,170 (0.0002)	P
Ptch1	1439663_at 5′	672 (0.0854)	Aa	369 (0.0811)	Pa
	1450824_at 3′	130 (0.5448)	A	24 (0.7566)	A
	1428853_at most 3′	10,091 (0.0002)	P	22,636 (0.0002)	P
Ptch2	1457256_x_at 5′	1,670 (0.0326)	P	3,938 (0.0004)	P
	1422655_at 3′	241 (0.3016)	A	1,632 (0.0081)	P
Gli1	1449058_at	528 (0.0745)	Aa	1,182 (0.0006)	P
Gli2	1459211_at	3,535 (0.0007)	P	2,815 (0.0004)	P
Gli3	1455154_at 5′	7,380 (0.0010)	P	5,342 (0.0006)	P
	1456067_at Middle	2,493 (0.0187)	P	2,540 (0.0056)	P
	1450525_at 3′	644 (0.1671)	Mc	231 (0.1360)	A
Fgf8a	1451882_a_at	15,621 (0.0010)	P	14,731 (0.0006)	P
En-1	1418618_at	3,179 (0.0002)	P	2,948 (0.0008)	P
Wnt7a	1423367_at	8,010 (0.0002)	P	6,311 (0.0002)	P
Hoxd11	1450584_at	82 (0.5196)	A	164 (0.1573)	A
Hoxd12	1450553_at	108 (0.5453)	A	62 (0.4346)	A

Figure 4. Sonic hedgehog (Shh) signaling components in the mouse limb ectoderm. A: Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of cDNA generated from stage 1 and stage 4 ectodermal hulls. B: RT-PCR products generated and electrophoresed on a 1.5% agarose gel. A,B: Template cDNA from stage 1 (A) and stage 4 (B). C: Section in situ hybridization of embryonic day (E) 11 mouse limb bud showing Ptch1 expression in the postaxial mesenchyme and overlying dorsal ectoderm (×100). AER is indicated by arrowhead. Boxed area is shown in inset at ×200. D: Immunohistochemistry section of an E10.5 mouse right forelimb bud sectioned across the dorsal–ventral axis. A, anterior; P, posterior. Shh is localized to the posterior mesenchyme and overlying ectoderm.

To determine where in the limb ectoderm Ptch1 is expressed, section in situ hybridization assays were performed on frozen cryostat sections of embryonic day (E) 11 limb buds. In addition to the mesenchymal domain of expression, a proximal posterior domain within the dorsal limb ectoderm was consistently detected (Fig. 4C). Other regions of the limb ectoderm, including the AER, were devoid of expression. The possibility that Shh directly signals to the ectoderm is supported by the immunohistochemical localization of Shh protein in not only the posterior mesenchyme corresponding to the ZPA but also in the overlying posterior limb ectoderm/AER (Fig. 4D).

DISCUSSION

Our previous studies on mouse embryos exposed during mid-gestation to teratogens that induce postaxial forelimb ectrodactyly revealed that drug-exposed postaxial limb mesenchyme expressed Shh but lacked “polarizing activity,” the ability to induce digit duplications when grafted to the anterior mesenchyme of a stage 20 chick wing bud (Bell et al., 1999; Scott et al., 2005). The data presented herein has further explored the inability of teratogen-exposed ZPA tissue to induce digit duplications. Using an in vitro Shh signaling assay in which the ability of limb bud cell lysates to activate a Gli-responsive promoter in Shh-LIGHT2 cells revealed that teratogen-exposed lysates exhibited reduced Shh signaling activity compared with controls. However, RT-PCR and Western blotting indicated that the diminished Shh signaling activity could not be attributed to differences in the levels of Shh transcription or translation within the teratogen-exposed tissue. These observations are in contrast to that observed in Wnt7a^−/− (Parr and McMahon, 1995; Adamska et al., 2004) and MHoxCre/Shh^floxed animals that also exhibit postaxial ectrodactyly but with detectable reductions in the level of Shh expression (Lewis et al., 2001).

The inability of teratogen-exposed ZPA tissue to induce a duplication is perplexing because teratogen-exposed ZPA tissue can induce many of the early events classically considered as part of a polarizing response, including expansion of the anterior chick wing margin, anterior AER expression of Fgf4, and ectopic domains of Ptch1, Bmp2, and Gli1. However, in contrast to control ZPA tissue, teratogen-exposed ZPA grafts were unable to respecify the developmental fate of cells normally destined to die within the ANZ of the host chick wing. Although previous studies have demonstrated that the duplicating effect of recombinant Shh on the anterior limb mesenchyme is dose and time dependent, a correlation between induction of ectopic anterior mesenchymal gene expression domains and duplicating activity has only been reported for Bmp7, Hoxd13, and Hoxd11(Yang et al., 1997). In this study, we have examined the ability of teratogen-exposed ZPA tissue to ectopically up-regulate anterior expression of Bmp2, Ptch1, Gli1, Gremlin, and Fgf4. We observed that, like control ZPA tissue, teratogen-exposed ZPA tissue could up-regulate anterior expression of Bmp2, Ptch1, Gli1, and Fgf4. To date, Bmp2, Ptch1, and Gli1 have only been demonstrated to be up-regulated after ectopic overexpression of either dHand or Shh (Laufer et al., 1994; Marigo et al., 1996a, b; Yang et al., 1997; Drossopoulou et al., 2000; Fernandez-Teran et al., 2000). Of interest, although misexpression of dHand consistently induced ectopic anterior domains of Gli1 and Ptch1, an ectopic Shh expression domain was not usually detected, suggesting that either undetectable low levels of Shh are required for the induction of these genes or that other signaling molecules can also induce their expression (Fernandez-Teran et al., 2000). The LIGHT2 cell assay suggests that teratogen-exposed ZPA tissue does possess a low level of Shh signaling activity. However, the observed expansion of the anterior wing margin, anterior AER, and induction of Fgf4 expression in response to teratogen-exposed ZPA tissue could be attributed to the presence of other signaling molecules within the grafted tissue such as Bmp2 or Gremlin. Bmp2-expressing cells do not induce ectopic Bmp2 expression domains in a host chick wing but can induce anterior AER expression of Fgf4, a growth response by the anterior mesenchyme, an ectopic Hoxd expression domain, and a low incidence of extra digit 2s (Duprez et al., 1996). Ectopic Gremlin expression can also induce alterations in Fgf4 expression and expansion of the anterior wing margin, without inducing digit duplications (Capdevilla et al., 1999).

Recent studies have suggested that Shh up-regulates expression of Gremlin, which then signals to the ectoderm to regulate formation/maintenance of the AER (Zuniga et al., 1999; Michos et al., 2004). Although teratogen-exposed ZPA tissue was unable to induce an ectopic anterior domain of Gremlin expression in the chick host, Gremlin expression within the mouse limb appeared normal in teratogen-exposed limb buds (data not shown). Furthermore, although the anterior and distal portions of the AER are maintained in teratogen-exposed limb buds, the dorsal and ventral boundaries are not smooth and the entire posterior portion of the AER is lost (Bell et al., 1999). These observations are in contrast to the inability of Shh and Gremlin mutants to maintain a functional AER (Zuniga et al., 1999; Sun et al., 2000; Chiang et al., 2001; Kraus et al., 2001; Khokha et al., 2003; Ros et al., 2003; Michos et al., 2004). Loss of the posterior AER margin in teratogen-exposed embryos is reminiscent of animals deficient in expression of the dorsal ectoderm marker Wnt7a (Parr and McMahon, 1995; Adamska et al., 2004). Based on these observations and others, it has been proposed that there is a feedback loop between the three limb axes where both the dorsal ectoderm and the AER are involved in the maintenance of Shh expression/the ZPA (Niswander, 2003). In return, Shh has been postulated to maintain the AER through Gremlin (Zuniga et al., 1999; Khokha et al., 2003). We suggest that Shh signaling may also maintain the posterior AER by directly signaling to the non-AER ectoderm overlying the ZPA. Data herein and previous immunohistochemistry studies localized high levels of Shh protein not only in the limb mesoderm but also in the overlying posterior ectoderm (Fig. 4D; Gritli-Linde et al., 2001). Furthermore, we have performed microchip array experiments on isolated ectodermal hulls from emerging stage 1.5 limb buds that had no detectable mesenchymal Shh expression and stage 4 limb buds with a prominent mesenchymal Shh domain and mature AER. Within the ectoderm, there is no evidence of Shh expression at either developmental stage, yet, there is an up-regulation of Ptch1, Ptch2, and Gli1 expression in the ectoderm of stage 4 limbs and high levels of Smo expression are detected at both stages. Quantitative RT-PCR and section in situ hybridization confirmed the presence of Ptch1 expression in the ectoderm, consistent with previous section in situ data indicating that, although Ptch1 expression is not detected in the AER, it is in the ectoderm overlying the ZPA (Hahn et al., 1996). Dogma would suggest that Ptch1 expression is restricted to the mesoderm; however, few section in situ data of Ptch1 in the limb are present within the literature and what are present are data acquired from whole-mount protocols, which may have destroyed the overlying ectoderm. Supportive of the argument that Shh signals directly to the ectoderm are recent studies using a Gli1-inducible CreER^T2 transgenic line to fate map cells responding to Shh signaling (Ahn and Joyner, 2004). These studies revealed that Shh signaling occurs in the posterior limb ectoderm and its positive aspects are mediated by Gli2. The limb ectoderm's responsiveness to Shh signaling is also indicated by the unexpected observation that Ptch1 is induced in the chick wing dorsal ectoderm in response to overexpression of a Gli-VP16 fusion protein containing the Gli zinc-finger DNA binding domain (Marigo et al., 1996b). Notably, although the fusion protein was expressed in the mesenchyme and ectoderm, ectopic Ptch1 was only observed in the ectoderm. Thus, it is plausible that teratogenic insult may interfere with the ability of Shh to directly signal to the ectoderm, resulting in a lack of proper formation of the posterior AER. Because recent fate mapping studies in the limb have revealed that the Shh-expressing cells give rise predominantly to the fourth and fifth digits (Harfe et al., 2004), the absence of the posterior AER in teratogen-exposed embryos would prohibit expansion of this population of precursor cells resulting in the observed malformations. The observation that teratogen exposure only induces these defects during the window when both the ZPA and AER are initially being formed suggests that regulation of this interrelationship occurs early in the ontogeny of both signaling centers, yet manifestation of the upset as detected by alteration in the expression of known molecular markers is delayed (Bell et al., 1999). A similar delay is seen in Shh−/− mutant limbs, where the AER precursors are properly initiated yet, in the absence of Shh, the AER begins to deteriorate (Sun et al., 2000; Chiang et al., 2001; Kraus et al., 2001; Ros et al., 2003). In the Shh−/− mouse embryo this begins by E10.5, the time at which teratogen-induced perturbations in the posterior margin of the AER become first evident. These results indicate that the components of the regulatory relationship between the ZPA, dorsal and ventral ectoderm, and the AER are not yet clearly defined and await further discovery.

It is currently unclear how teratogen-exposure disrupts Shh signaling. RT-PCR and Western blot analysis suggest that Shh transcription and translation are not disrupted by teratogen-exposure. Other putative sites of teratogen interference include (1) improper posttranslational processing of Shh, (2) a failure of mature Shh to be secreted from the cell, or (3) the induction or activation of a Shh signaling inhibitor by teratogen exposure. Posttranslational processing of the 45-kDa Shh protein includes an autocleavage event to yield the 19-kDa ShhN fragment responsible for signaling. This fragment is then modified by the addition of cholesterol to its new C-terminus and palmitate to the N-terminus (for review, see Ingham and McMahon, 2001). We believe that teratogen exposure does not alter normal proteolytic cleavage of Shh because this is the predominant form detected by the Western blot analysis. Furthermore, the genetic abolition of cholesterol or palmitate addition yields a limb phenotype with missing central digits, not the phenotype postaxial ectrodactyly associated with teratogen exposure (Lewis et al., 2001; Chen et al., 2004). Alternatively, reduced Shh signaling by grafted teratogen-exposed ZPA tissue could be explained by alterations in Shh secretion thought to be facilitated by Dispatched (Ingham and McMahon, 2001). This protein, as well as the Shh receptor Ptch, belong to a family of RND permeases (Tseng et al., 1999). These proteins function in prokaryotes as proton-driven efflux pumps to secrete drugs, heavy metals, or endogenous molecules (Nies, 1995; Tseng et al., 1999). This function is a potential site of teratogen perturbation because acetazolamide does disrupt acid/base balance and has been shown to lower pH in the mouse embryo limb bud (Schreiner et al., 1995). However, this explanation also is not completely satisfactory, because a teratogen-induced disruption in Shh release from expressing cells should be overcome in the LIGHT2 assays using cell lysates, yet these assays also indicate little Shh signaling activity. Teratogen exposure could also affect Shh multimerization (Zeng et al., 2001); however, the site, mechanism, and functional importance of multimerization have not yet been elucidated.

An alternative possibility is that teratogen-exposure induces or activates an inhibitor of Shh signaling. Schaller and Muneoka (2001) previously suggested the existence of a polarizing activity inhibitor normally made in the anterior mesenchyme that interacts with heparin sulfate moieties in the extracellular matrix. Gas-1 is normally expressed in the anterior mesenchyme and can act as an inhibitor of Shh activity (Lee, 2001; Liu et al., 2002). Hedgehog interacting protein was identified as an inhibitor of Shh activity expressed in the posterior limb mesenchyme; however, its putative alteration by teratogen exposure is not known (Chuang and McMahon, 1999). Favoring the existence of an inhibitor is our previous result that, upon recombining teratogen-exposed mesoderm with control ectoderm for only 2 hr, polarizing activity was restored, but only if the normal anterior–posterior polarity of the ectoderm and mesoderm were maintained. These observations suggest that the postaxial ectoderm produces a factor that can relieve the teratogen-induced inhibitory effect on “polarizing activity.”

In summary, we have shown that teratogen exposure leading to postaxial forelimb ectrodactyly is accompanied by lowered Shh signaling. The level of signaling, approximately 25% of normal, is sufficient to up-regulate many of the processes that are needed for polarizing activity but is insufficient to respecify the cell death program in anterior limb mesenchyme cells. We also provide evidence indicating that Shh may signal directly to the limb bud ectoderm and suggest that this direct signaling is required for maintenance of the posterior AER.

EXPERIMENTAL PROCEDURES

Animals

Studies were conducted in C57BL/6CrIBR mice purchased from Charles River. Individual males were placed in female cages for the last 1–2 hr of the 12-hr dark cycle. The presence of a vaginal plug indicated a successful mating and 9:00 AM was considered time 0 of pregnancy.

Control animals included both untreated animals and those administered the vehicles (carboxymethyl cellulose or oil). Vehicle control and drug-treated animals were lightly anesthetized before receiving subcutaneous injections on E9.5. For the chick grafting and Shh-LIGHT2 cell assays, acetazolamide (gift from Lederle Laboratories) was suspended in 0.3% carboxymethyl cellulose at a concentration of 25 mg/ml and delivered at a dose of 250 mg/kg. Two hours later, a solution of 0.05 mg/ml benzamil-HCl (Research Biochemicals International, Natick, MA) in water was administered at a dose of 0.5 mg/kg. For PCR and Western blot analysis, animals were intraperitoneally administered 400 mg/ml acetazolamide (Sigma) suspended in vegetable oil. This dosing regimen also yields a >80% incidence of postaxial mouse forelimb defects as was previously observed with acetazolamide suspended in carboxymethylcellulose (Bell et al., 1999). On E10–E11, embryos were collected and the limb buds were excised for determining either Shh content or signaling activity.

In Situ Hybridization

Whole-mount assays were performed essentially as described by Wilkinson and Nieto (1993) with proteinase K treatments as described in (Bell et al., 1998). For sections, embryos were fixed overnight in 4% paraformaldehyde and equilibrated in 30% sucrose before being embedded in OTC. Fourteen-micron sections were generated on a cryostat and processed using the protocol of Torenson et al. (1999). Slides were developed for 2 days in BMP-Purple (Roche) in the presence of 0.5 mg/ml Levamisole (Sigma). Digoxigenin-labeled riboprobes were transcribed from linearized DNA templates using either SP6, T3, or T7 RNA polymerase. Template DNAs were generously provided by Andrew McMahon (mouse Shh), Matthew Scott (mouse Ptch1), Lee Niswander (chick Bmp2, Bmp4, and Fgf4), Alexandra Joyner (mouse Gli3), Richard Harland (chick Gremlin), William Upholt (chick Msx2), and Cliff Tabin (chick Ptch1, Gli, and Gli3). Embryos were examined and photographed by using a Wild M5A stereoscope.

Limb Measurements

Limbs were dissected 30 hr after receiving a ZPA graft such that the limb body wall junction was retained. Limbs were then photographed from the ventral side at 57×. Using 4 × 6 prints, the junction of the limb with the body wall was demarcated. The limb length was determined by measuring from the middle of the limb/body wall junction to the tip of the limb at a point giving the greatest length across the proximal–distal axis. The width of the limb was determined by measuring in 25 mm from the limb tip along the limb length line and drawing an intersecting line 60 degrees toward the anterior margin of the limb. Width was then measured between the intersection of this line and the anterior and posterior limb margins (Fig. 1E–H). Measured values were only corrected for microscope magnification and, therefore, are not absolute measurements.

Shh Activity Assay

For each sample of cell extract, a litter of E10.5 implantation sites from either untreated embryos or embryos exposed to acetazolamide on E9.5 were removed, immediately frozen in liquid nitrogen, and stored at −80°C until use. Immediately before assay, embryos were thawed and dissected free of their surrounding membranes. Stage 3–3.5 (Wanek et al., 1989) right forelimbs were pooled in an Eppendorf tube. The excess dissection fluid was removed and replaced with 200 μl of serum-free DMEM. Samples were sonicated and DNA in the lysates quantitated using a Hoeffer DyNA Quant 200 Fluorimeter. Lysates were diluted in DMEM (0.5% fetal bovine serum [FBS]) to a concentration of either 50 or 25 ng/ml of DNA in a final total volume of 100 μl. Shh-LIGHT2 cells (Taipale et al., 2000) were cultured in DMEM containing 10% FBS in a 96-well plate (2.5 × 10⁴ cells/well) for approximately 24 hr (confluency). The medium was removed from duplicate wells and replaced with 100 μl of diluted limb bud cell lysate. To serve as a background control, a duplicate set of wells received only 100 μl of DMEM (0.5% FBS). Cells were incubated for 24 hr, and luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized to cellular Renilla activity in each lysate.

Real Time RT-PCR

Precise levels of message expression of Shh, Ptch1, and Gli3 were determined by RT-PCR. The Stratagene Nanoprep RNA kit was used to isolate total RNA from either entire right forelimb buds or ectodermal hulls isolated as described in the chip array experiments. Equal amounts of total RNA were primed with Oligo dT and reverse transcribed using Superscript II (Invitrogen). Samples were PCR amplified in the presence of SyberGreen (Quantitect Sybergreen PCR kit, Qiagen) using gene-specific primers for Shh, Ptch1, Gli3, and α-Tubulin. Chip array data and preliminary experiments indicated that α-tubulin mRNA levels were unchanged by acetazolamide exposure and, thus, was used to normalize the quantitative PCR data. The PCR amplifications and monitoring of product generation per cycle were performed using a DNA Engine Opticon 2 Continuous Fluorescence Detector and evaluated using its software. Gene expression levels were determined by gene-specific standard curves in the linear amplification phase. All values were normalized to tubulin within the same sample. Primers for Shh, Ptch1, and Gli3 spanned one or more introns to ensure the amplification of mRNA and not contaminating DNA. The gene-specific primer pairs used were the Shh primers 5′-GCTGACCCCTTTAGCCTACAAG-3′ and 5′-CCACGGAGTTCTCTGCTTTCAC-3′, the Ptch1 primers 5′-CCCCAAACTCCACTCA- AAAGG-3′ and 5′-CACCAACACCAAGAGCAAGAAAC-3′, the Gli3 primers 5′-GGAGCATTTGGTGAGCAGAAGG- 3′ and 5′-AGGGGCTTTGGAGGGTAGAATAGG-3′, and the α-tubulin primers 5′-CTGTATGAAAGCACACATTGCCAC-3′ and 5′-ATGGAGGAGGGT- GAGTTCTCTGAG-3′.

Western Blot Analysis

Forelimb buds from E10.5 vehicle or acetazolamide-exposed embryos were excised and cut in half, and the posterior half was collected for Western blot analysis. Pooled limb bud samples were lysed in RIPA buffer in the presence of a protease inhibitor cocktail and then homogenized by passage through a narrow gauge needle followed by water bath sonication. Samples were centrifuged at 2,000 × g for 5 min. Laemmli buffer (5×) was added to the samples before boiling for 10 min. Samples were then run on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose. After probing the membranes for Shh with poly-clonal antibody H-160 (Santa Cruz), the membranes were stripped and reprobed using a monoclonal antibody to tubulin (β-tubulin mouse monoclonal, Sigma, TUB 2.1).

ZPA Grafts

Before grafting, fertilized White Leghorn eggs (SPAFAS) were incubated at 38°C for 3.5 days. During the third day of incubation, 1–2 ml of albumin was removed, eggs were windowed, and 3 drops of penicillin/streptomycin (50 units/ml) were added. Donor pieces of mouse ZPA tissue were isolated and grafted between the AER and anterior mesenchyme of stage 19/20 (Hamburger and Hamilton, 1951) chick wing buds. The mouse donor tissue was isolated from the Shh expressing, posterior, distal margin of right forelimb buds (stages 3–4; Wanek et al., 1989) from drug-treated and control embryos. After grafting, windows were taped closed, and eggs were returned to the incubator for an additional 24, 30, 36, or 42 hr.

Nile Blue Staining

For cell death studies, chick embryos were dissected free of their surrounding membranes and placed into Nile blue sulfate diluted in phosphate buffered saline (PBS) 1:20,000 for 15–20 min at room temperature. Embryos were destained in PBS (−Ca²⁺, −Mg²⁺) for 20–30 min and immediately photographed.

MicroArray Experiments

Stage 1.5 and Stage 4 limb buds were excised from the body wall. To isolate ectodermal hulls, limbs were trypsizined (stage 1.5 limbs for 30 min and stage 4 limbs for 50 min) followed by a 30-min incubation in serum. Ectoderm was removed from the underlying mesenchyme using watchmaker forceps, rinsed in PBS, and pooled. Total RNA was isolated using the Stratagene Nanoprep RNA isolation kit and quantitated using Ribogreen (Molecular Probes). Thirty stage 1.5 limb buds yielded approximately 2 μg, whereas, 24 stage 4 limbs yielded ∼5 μg of total RNA. Samples were given to the Cincinnati Children's Hospital MicroArray Core for amplification and hybridization. The Amersham Codelink kit was used to amplify and label 500-ng samples. Labeled product was hybridized to the Affymetrix mouse genome 430v2.0 chip containing most of the mouse transcriptome. Three independent biological samples were prepared for each stage. Expression values were obtained using the Affymetrix GeneChip Operating Software. The target signals of each chip were scaled to 1,500 using all probe sets. Expression values and P values are the average of the three chips.

Immunohistochemistry

E10 and E10.5 embryos were fixed overnight in Sainte Marie's fixative (95% ethanol, 1% acetic acid) at 4°C. The following morning, embryos were embedded in paraffin and sectioned at 10 μm. Within the same week, slides were processed for Shh localization as described by (Kawakami et al., 2002). Anti-Shh polyclonal antibody (H-160; Santa Cruz) was used diluted 1:800 in PBS. The secondary antibody, biotinylated swine anti-rabbit IgG (Dako Cytomation, Denmark) was used at 1:200. The signal was amplified using the Tyramide amplification kit (Perkin Elmer). Slides were counterstained with 0.5% toluidine blue, 10 mM sodium acetate, pH 4.6.