Manipulations of PKA in chick limb development reveal roles in digit patterning including a positive role in Sonic Hedgehog signaling

Developmental Biology 305 (2007) 312 – 324 www.elsevier.com/locate/ydbio

Manipulations of PKA in chick limb development reveal roles in digit patterning including a positive role in Sonic Hedgehog signaling Eva Tiecke a,1 , Roisin Turner b , Juan Jose Sanz-Ezquerro a,2 , Anne Warner b , Cheryll Tickle a,⁎ a

Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK b Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, UK Received for publication 27 September 2006; revised 9 February 2007; accepted 13 February 2007 Available online 21 February 2007

Abstract Sonic Hedgehog (Shh) signaling by the polarizing region, at the posterior of the vertebrate limb bud, is pivotal in determining digit number and identity. Shh establishes a gradient of the bifunctional transcriptional effector, Gli3, with high levels of full-length activator (Gli3A) in the posterior bud, where digits form, and high levels of shorter repressor (Gli3R) in the anterior. Repressor formation depends on protein kinase A (PKA), but in Drosophila, PKA also plays a role in activator function. Increasing PKA levels in chick limb development using Forskolin had no effect on posterior polarizing activity but weak polarizing activity, based on ligand-independent Shh signaling, was induced in anterior limb bud cells resulting in extra digits. Manipulating PKA activity levels directly with a retrovirus expressing activated PKA induced extra digits similar to those induced by Forskolin treatment suggesting that PKA may have a previously unrecognized positive role in Shh signaling in vertebrate limbs. Expressing dominant negative PKA also induced extra, sometimes multiple digits, from anterior limb bud demonstrating the negative role in Shh signaling. PKA levels in the limb bud are high posteriorly and low anteriorly, suggesting that PKA activity may influence the outcome of Shh signaling in normal development. © 2007 Elsevier Inc. All rights reserved. Keywords: Chick; Limb; Sonic hedgehog; Gli3; PKA

Introduction A key question in embryonic development is how different parts of the body arise in the proper places. A good model to address this question is the digit pattern in vertebrate limbs, which is controlled by signaling of the polarizing region. The extracellular signaling molecule, Sonic Hedgehog (Shh), is now known to be pivotal in this process (Riddle et al., 1993) and considerable progress has been made in dissecting out the Hedgehog (Hh) signal transduction pathway in vertebrates (reviewed by Cohen, 2003; Hooper and Scott, 2005). Mutations in a growing number of genes that encode components of the pathway, whose functions, in many cases, are not yet well⁎ Corresponding author. E-mail address: [email protected] (C. Tickle). 1 Current address: Developmental Genetics DKBW Centre for Biomedicine, Mattenstrasse 28, Ch-4058 Basel, Switzerland. 2 Current address: Departmento de Immunologia y Oncologia, Centro Nacional de Biotechnologia, Campus de Cantoblanco–UAM, 28049 Madrid, Spain. 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.02.017

understood, lead to changes in digit number and pattern, for example the ta3 gene (Davey et al., 2006) and genes that encode intraflagellar proteins (Huangfu et al., 2003; Liu et al., 2005). Therefore, it is becoming increasingly important to dissect out the pathway in detail and understand how it is regulated. Here, we examine the role of protein kinase A (PKA), which has been implicated in modulating several steps in Hh signal transduction, in patterning the digits of the chick wing. The polarizing region is a region of mesenchyme cells at the posterior margin of the limb bud and its signaling properties were discovered through its ability to induce extra digits (polarizing activity) when grafted to the anterior margin of a second wing bud (Saunders and Gasseling, 1968). Following a polarizing region graft, an extra set of digits 432 arise from the anterior part of the wing bud and are arranged in mirror-image symmetry with the normal set 234. Signaling has been shown to be dose-dependent with, for example, grafts of small numbers of polarizing region cells inducing only an extra digit 2, and increasing numbers resulting in an additional digit 3 and finally an additional digit 4 (Tickle, 1981). Similar effects can be

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produced by varying the length of exposure to a polarizing region signal (Smith, 1980) and it has been suggested that cells first form an anterior digit and then are promoted over time to form more posterior digits (Eichele et al., 1985). Shh is one of three vertebrate Hedgehog genes and is expressed in the polarizing region (Riddle et al., 1993). Shh has been shown to diffuse across the posterior part of the limb bud (Gritli-Linde et al., 2001; Zeng et al., 2001). Application of Shh to the anterior margin of chick limb buds induces digit duplications in a dose- and time-dependent fashion (Yang et al., 1997). Shh binds to the receptor patched (Ptc) on responding cells, which allows signaling by the transmembrane protein smoothened (Smo) through a signaling complex containing the Gli family of transcriptional effectors, Gli1, Gli2 and Gli3 (reviewed Cohen, 2003; Hooper and Scott, 2005). In the presence of Hh ligand, full-length Gli protein (Gli3Fl) becomes converted through an unknown process into a transcriptional activator (GliA) (Chen et al., 1999), which translocates to the nucleus and activates expression of target genes. In the absence of Hh ligand, Gli proteins, in particular Gli3, are processed to a shorter form (GliR), which can also translocate to the nucleus but acts as a transcriptional repressor (reviewed Koebernick and Pieler, 2002). In normal chick limb buds, Gli3A is high in posterior and low in anterior regions whereas Gli3R is high in anterior and low in posterior regions (Wang et al., 2000). Analysis of Shh−/−, Gli3−/− and Gli3−/−; Shh−/− mouse embryos has shown that the main function of Shh in the limb, is to relieve Gli3 mediated repression of genes required for digit patterning. This relief of Gli3R leads to formation of a series of patterned digits from the posterior part of the limb bud while the anterior part, where Gli3R persists, does not give rise to digits (Litingtung et al., 2002; te Welscher et al., 2002). Proper functioning of the Hh pathway is known to involve several kinases, for example, GSK3, CK1, PKA and ubiquitin ligase, that modify components so that they transduce the Hh signal (or lack of Hh signal) properly (Hooper and Scott, 2005). In several vertebrate systems, PKA has been shown to be an antagonist of hedgehog signaling (Epstein et al., 1996; Hammerschmidt et al., 1996; Ungar and Moon, 1996) and this has been ascribed to its role in regulating processing of the Gli3 protein to the repressor form (Wang et al., 2000). Here we investigate the role of PKA in modulating the output of Shh signaling pathway in chick limb development both by applying the drug Forskolin and by overexpressing constitutively active and dominant negative PKA using the RCAS (Replication Competent Avian Sarcoma) viral system. Forskolin increases adenylate cyclase activity thereby increasing intracellular cyclic AMP and thus PKA activity. Forskolin has been shown in several developing systems including somite, brain, feather bud, tooth and testis (Fan et al., 1995; Hynes et al., 1995; Noveen et al., 1996; Cobourne et al., 2001; Yao and Capel, 2002) to inhibit Shh signaling. We showed that Forskolin treatment had no effect on polarizing region signaling but instead, surprisingly, induced anterior limb mesenchyme to form digits. We further showed that manipulating levels of PKA directly also resulted in the formation of extra digits.

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Materials and methods Embryos Fertilized White Leghorn chicken eggs were obtained from Henry Stewart (Lincolnshire), fertilized quail eggs from the Roslin Institute (Edinburgh) and standard pathogen-free (spf) fertilized eggs from Lohmann Tierzucht (Germany). All eggs were incubated at 37 °C and staged according to Hamburger and Hamilton (1951).

Micromass culture Micromass cultures were prepared from chick or quail limb buds (stages 20–21; Vogel and Tickle, 1993; Makarenkova et al., 1997). A 10 μl drop containing 4 × 104 cells/ml or 2 × 107 cells/ml was placed into the center of each well of a 4-well dish (Nunc). After 1 h, 500 μl of culture medium (F12/DMEM, 50/50; Gibco BRL with 10% FCS, 1% glutamine and 1% antibiotic/ antimycotic) was added and cultures were maintained for 24 h in a 37 °C incubator gassed with 5% CO2. Forskolin (Sigma) dissolved in DMSO was added to cultures at a final concentration of 100 μM.

Grafts of micromass culture and carriers soaked in Forskolin After 24 h, micromass cultures were scraped off the dish, cut into 8 pieces and grafted under a loop made by lifting away the apical ectodermal ridge at the anterior margin of a stage 20–21 chick limb buds (Fig. 1A). Various carriers, including heparin beads (Sigma), Affigel blue beads (Biorad), nitrocellulose with 2 different pore sizes (1.2 μm and 8 μm respectively, Millipore) and DEAE paper (Whatmann; Tickle et al., 1982), were soaked in 50, 100, 250 and 500 μM Forskolin for 1–2 h at room temperature and then grafted to the anterior margin host limb.

Alcian green staining Embryos were fixed in 5% trichloroacetic acid at day 9 of incubation, stained for cartilage using 0.1% Alcian Green in 70% acetic acid, dehydrated in ethanol and cleared in methylsalicylate.

QCPN staining Wings of chicken embryos with quail cell grafts were fixed at 9 days of incubation in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), embedded in O.C.T. compound (Miles), frozen and sectioned at 10 μm. Sections were stained as previously described (Tanaka et al., 2000) except that the secondary antibody was FITC anti-mouse (Sigma). Slides mounted in Citifluor (Agar Scientific) were viewed with a fluorescence compound microscope.

RT-PCR RNA was isolated from tissue using a QiaShredder column and Quiagen RNA Easy kit (Quiagen). cDNA was synthesized using AMV Reverse Transcriptase Kit (Roche) using oligo dT primers to select for mRNA only. The RT-PCR was carried out using the following primers. Chick Shh: forward 5′-AGAGAGACAGCCAGGGATAGG-3′ and reverse 5′-TGCCACTGAGTTTTCTGCTTT-3′, chick Gli3 :forward 5′-GACGGCAGGTATTAGTTCAAGG-3′ and reverse 5′-GCGTTCCTTCTCTCTCTGTGAT-3′, chick GAPDH: 5′-AGCTTACTGGAATGGCTTTCC-3′ and 5′-ACCATCAAGTCCACAACACGGTTGCTG-3′

In situ hybridization In situ hybridization was performed as previously described (Wilkinson and Nieto, 1993) with probes synthesized from the following templates: Shh (Roelink et al., 1994), Ihh (Vortkamp et al., 1996), dHAND (Fernandez-Teran

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E. Tiecke et al. / Developmental Biology 305 (2007) 312–324 A, leupeptin and chymostatin (Peptide Inc.) and 100 μg/ml PMSF (Sigma)). Protein concentration was assessed using a Bradford assay (Pierce, Coomassie Protein Assay Reagent Kit). 100 μg protein was loaded per well. Membranes were blotted with anti-Gli3 (1:500; Santa Cruz) and anti-rabbit HRP (1:10,000; Sigma) or anti-α-tubulin (1:50; Developmental Studies Hybridoma Bank, Iowa) and anti-mouse HRP (1:5000; Sigma). Results of the Western blots were quantified using AIDA Image Analyzer ver3.28 and a Student's T-test was performed on the data.

Radioactive PKA activity assay Micromass cultures or limb buds were lysed in buffer: 50 mM Tris–HCl pH 7.4, 0.27 M sucrose, 1 mM Na Ortho-vanadate pH 10, 1 mM EDTA, 1 mM EGTA, 10 mM Na B glycerophosphate, 10 mM NaF, 5 mM sodium pyrophosphate, 1% triton X-100. One inhibitor cocktail tablet (Complete, EDTA-free, mini, Roche, 1836153) and 50 μl β-mercaptoethanol were added freshly per 50 ml. PKA activity was monitored by transfer of 32PO3 from Isoblue [32P]-γ-ATP (ICN, 38101X.2) to the substrate peptide. The reaction contains 50 mM MOPS, pH 7.0, 5 mM MgCl2, 100 μM Kemptide, 100 μM [32P]-γ-ATP; 4500 Ci/mmol specific activity and tissue. An 8-min reaction was performed at 37 °C and, after washing, measured in a scintillation counter for 2 min without scintillant. Assays were conducted in triplicate and means calculated.

Virus infection RCAS viruses containing activated PKA (RCAS-aPKA), dominant negative PKA (RCAS-dnPKA; both a kind gift from Dr. A. Münsterberg) or RCAS-eGPF (cloned by Dr. E. Farrell) were obtained. aPKA contains an active mutant of catalytic subunit C of PKA whereas in dnPKA the cyclic AMP binding sites of the regulatory subunit have been mutated (Mellon et al., 1989). DF1 cells were transfected with DNA for these viruses. Medium was harvested and virus concentrated to give a minimal titer of 1 × 108 virus particles/ml. Chick embryos were infected by injecting concentrated virus into the coelom at stages 13–14 on the right hand side.

Results Fig. 1. Effects on chick wing pattern of grafts of either anterior or posterior limb bud cells cultured in micromass for 24 h with or without Forskolin. (A) Experimental protocol. (B–F) Whole mounts of 9- to 10-day chick wings, stained with Alcian Green. (B) Graft of control culture of posterior limb bud cells, digit pattern ?3234. (C) Forskolin-treated graft of posterior limb bud cells, digit pattern 24334, note extra element in lower arm. (D) Graft of control culture of anterior limb bud cells, normal wing skeleton, digit pattern 234. (E, F) Grafts of Forskolin-treated cultures of anterior limb bud cells (E) digit pattern 2234, note absent radius; (F) digit pattern 334, note reduced radius. (G) Digit pattern 2234 obtained after a graft of Forskolin-treated cultures of anterior limb bud cells plated at a density of 4 × 104/10 μl. (H, I) Grafts of DEAE paper soaked in 100 μM Forskolin. Digit patterns 2234 (H) and (33)4 (I).

et al., 2000), Bmp2 (Francis et al., 1994) Fgf4 (Niswander and Martin, 1992), Fgf8 (Crossley et al., 1996), Gremlin (Merino et al., 1999), Gli1 (Marigo et al., 1996a), Hoxa13 (Nelson et al., 1996), Ptc (Marigo et al., 1996b). The following numbers of embryos were hybridized 24 h after grafting micromass cultures. Shh and Fgf8: anterior control (AC) 9; anterior Forskolin treated (AF) 16; posterior Forskolin treated (PF) 5. Fgf4, Ptc and Gli1: AC 2; AF 4; posterior control (PC) 2; PF 2. Gremlin: AC 4; AF 6; PC 4; PF 4. Hoxa13: AC 2; AF 3; PC 1; PF 2. dHAND and Bmp2: 4 for each condition.

Western blot Micromass cultures or limb buds were isolated in RIPA buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with freshly added inhibitors (10 μg/ml pepstatin

Effect of Forskolin on anterior mesenchyme cells In order to explore the role of PKA in signaling of the polarizing region, we treated micromass cultures of quail posterior limb mesenchyme cells with Forskolin, then grafted them to the anterior margin of host chick wing buds (Fig. 1A). We monitored resultant effects on digit pattern in 10-day wings. Untreated posterior limb mesenchyme cell cultures were grafted for comparison. We also made micromass cultures from anterior limb mesenchyme and treated them with Forskolin and grafted both treated and untreated cultures to host wing buds (Fig. 1A). Table 1 shows the data on digit pattern and method for calculating polarizing activity. Forskolin had no clear effect on the polarizing activity of cultured posterior limb mesenchyme cells. Wings with grafts of either untreated (Fig. 1B, n = 17) or treated posterior limb mesenchyme cultures (Fig. 1C, n = 22) almost always had digit duplications (16/17 and 21/22 respectively) and polarizing activity was 53% and 61% respectively. Duplications that included a posterior digit 4 were frequent. In some host wings, the radius was absent or partially formed and occasionally the distal end of the humerus was enlarged (4/17 for grafts of untreated posterior limb mesenchyme cultures; 13/22 for Forskolin-treated posterior limb mesenchyme cultures). Extra digits were not always associated with defects in fore- or upper arm. One wing

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Table 1 Effects on host chick wing digit pattern of grafting anterior or posterior quail limb micromass cultures treated for 24 h with or without Forskolin Treatment

Posterior control Posterior Forskolin Anterior control ⁎ Anterior Forskolin ⁎

n

17 22 17 35 47 36

Total number of digits formed

Type of extra digits obtained

Polarizing Wings with activity extra digits

3

4

5

Mean digit Normal digit Additional Additional Additional or Additional number pattern 234 blip 2 transformed 3 4

(%)

(%)

6 6 17 35 36 28

8 12 0 0 11 8

3 4 0 0 0 0

3.8 3.9 3 3 3.2 3.1

53 61.2 0.0 0 14.4 5.6

94 95 0 0 47 22

1 1 17 35 25 28

0 0 0 0 4 0

2 2 0 0 11 8

11 12 0 0 7 0

3 7 0 0 0 0

All grafts were from micromasses in which 2 × 105 cells/10 μl were plated apart from rows indicated by ⁎ in which 4 × 104 cells/10 μl were plated. Bifurcated digits were counted as 1. Additional blip includes b234 and bb234. Additional 2 include b2234, (22)34, 2234. Additional 3 include 32(34), 3234, 334, 3334, 3b34, ?3234, (b3)234, 3(22)34, 32234, (33)234, b32234. Additional 4 include 24334, 34.334, 4334, 43b34, (43)234, 43(22)34, 43234. ðdigit 4Þ  4 þ ðdigit 3Þ  2 þ ðdigit 2Þ  1 þ ðblipÞ  0:5  100 ¼ polarizing activity total number of wings 4

grafted with a Forskolin-treated posterior limb mesenchyme culture had an extra element in the forearm (Fig. 1C). Wings with grafts of untreated cultures of anterior limb mesenchyme cultures never had extra digits (Fig. 1D, quail to chick n = 17; chick to chick n = 35) although some had a radius defect. Unexpectedly, 47% of chick wings grafted with cultured quail anterior limb mesenchyme cells treated with Forskolin had changes in digit pattern (n = 22/47, Figs. 1E, F) while 28% of wings with grafts of Forskolin-treated chick anterior limb mesenchyme cultures also showed changes (8/28; Fig. 1G). In both sets of experiments, the change in digit pattern consisted, nearly always, of an extra anterior digit 2 (Fig. 1E), i.e., duplication of the most anterior digit; sometimes a digit 2 was replaced by a digit 3 (Fig. 1F). Defects in more proximal elements, mainly a missing or reduced radius, were observed in some cases. Chick anterior limb mesenchyme cells cultured at lower cell densities (10 μl of 4 × 104 cells/ml) also induced extra digits when treated with Forskolin (5/15), while grafts of cells from untreated anterior limb mesenchyme cultures at the same cell densities had no effect on wing patterning (10/10). Thus treatment of cultured quail or chick anterior limb mesenchyme cells with Forskolin unexpectedly results in formation of an extra digit 2 or replacement of digit 2 by a digit 3 when these cells are grafted back into host limbs. To investigate whether the extra or altered digit arises from the Forskolin-treated anterior limb mesenchyme cells or from host tissue, we studied the fate of cultured quail anterior limb mesenchyme cells treated with Forskolin grafted to chick limb buds. Graft quail and host chick cells can be distinguished using the QCPN antibody. The extra anterior digit (digit 2; Figs. 2A′, A″) consisted of both quail and chick cells (Fig. 2A‴, n = 3). Graft cells were found in host mesenchyme along the anterior edge and at the base of the extra digit (Fig. 2A‴, arrows). They were also found more proximally in ectopic feather buds (Fig. 2A‴, arrowheads) and cartilage. For comparison, we examined distribution of quail cells from cultures of posterior limb

mesenchyme treated with Forskolin when grafted to a host chick wing. In this case, the extra digit, a digit 3 (Fig. 2B′, B″), also contained both graft and host cells. Graft cells were found in host mesenchyme along the anterior margin of the extra digit, as with grafts of Forskolin-treated anterior limb mesenchyme cells, but extended more distally (Fig. 2B‴; arrows). No quail cells were detected in the cartilage of the extra digit but there were quail cells in more proximal cartilage elements of the wing (data not shown). We conclude that Forskolin-treated anterior limb mesenchyme cells entrain host anterior limb mesenchyme cells to participate in extra digit formation and behave similarly to Forskolin-treated posterior cultured cells or polarizing region cells (CT unpublished). We then tested whether applying Forskolin directly to the anterior margin of chick limb buds on inert carriers could lead to formation of extra anterior digits or transform identity of the anterior digit. With most of the carriers, no extra digits formed (124/161 cases) but, with Forskolin soaked pieces of DEAE paper (Whatmann), similar digit duplications were induced in host wing buds to those produced by grafts of Forskolin-treated micromasses of anterior limb mesenchyme cells in 10/37 cases. Either an extra digit 2 (Fig. 1H) was induced or a digit 3 formed in place of a digit 2 (Fig. 1I). The optimal concentration of Forskolin was 100 μM (Table 2), the concentration used to treat micromasses. Both methods gave roughly the same polarizing activity. Thus either treating anterior limb mesenchyme cultures with Forskolin or treating anterior limb buds directly with Forskolin leads to additional digit formation or promotes a digit 2 to a digit 3. Gene expression in anterior limb mesenchyme after Forskolin treatment The digit pattern change produced by grafts of Forskolintreated anterior limb mesenchyme cultures could be explained by the induction of Shh expression in the cells. However, we

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Fig. 2. QCPN staining to study distribution of quail graft and chick host cells in extra digits and RT-PCR to examine Shh expression in cultured cells. (A′) Host chick wing grafted with Forskolin-treated culture of quail anterior limb bud cells; digit pattern 2234. (A″) High power of panel A′ showing extra digit 2. (A‴) Frozen section through extra digit showing labeled quail cells along anterior edge and in ectopic feather buds. (B′) Host chick wing grafted with Forskolin-treated quail posterior limb bud cells; digit pattern 3234. (B″) High power of panel B′ showing extra digit 3. (B‴) Frozen section through extra digit 3 showing labeled quail cells along anterior edge (C) RT-PCR for Shh in micromass cultures at 1 and 24 h and in stage 20 limb bud. (C′) RT-PCR for GAPDH in same samples as control. AC = untreated anterior mesenchyme culture, AF = Forskolin-treated anterior mesenchyme culture, PC = untreated posterior mesenchyme culture, PF = Forskolin-treated posterior mesenchyme culture, A = anterior limb bud, P = posterior limb bud.

could detect no Shh expression by RT-PCR in Forskolin-treated cultures of anterior limb mesenchyme cells after either 1 or 24 h of culture (Fig. 2C) as was the case with untreated cultures of anterior limb mesenchyme cells. Treated and untreated cultures of posterior limb mesenchyme cells expressed Shh at 1 h as expected but showed no detectable expression of Shh at 24 h consistent with previous reports (Kimura and Ide, 1998). GAPDH was used as a positive control (Fig. 2C′). Even though no Shh expression could be detected in the Forskolin-treated micromasses of anterior limb mesenchyme cells, Shh expression might be switched on when the anterior limb mesenchyme cell micromasses are placed under the apical ridge in a host wing bud. Therefore, we examined Shh expression 24 h after grafting treated and untreated cultures of both anterior and posterior limb mesenchyme into host limb buds by in situ hybridization. However, Shh expression could not be detected in any of these grafts (Figs. 3A–C). There have been reports of redundancy between the different vertebrate Hhs (Yang et al., 1998; Pathi et al., 2001), and Ihh is known to be expressed during chondrogenesis (Vortkamp et al., 1996;

Koyama et al., 1996). We therefore examined Ihh expression in grafted cultures but no expression could be found after 24 h (data not shown). We conclude that the effect of Forskolin on anterior limb mesenchyme cells is not dependent on induction of Shh or Ihh expression. One hypothesis that would explain our observations is that Forskolin activates the Shh signaling pathway in anterior limb mesenchyme in the absence of ligand. This hypothesis was tested by examining, in host limb buds grafted with Forskolintreated anterior limb mesenchyme cells, expression patterns of genes known to be read outs of Shh signaling and associated with digit formation. Gene expression was compared with that in limb buds grafted with untreated anterior limb mesenchyme cells and with untreated and treated posterior limb mesenchyme cells. We examined expression of genes, Ptc and Gli1, known to be activated by Shh and also expression of genes which more likely depend on repression by Gli3R, Hoxa13, Gremlin, dHAND and Bmp2 (Litingtung et al., 2002; te Welscher et al., 2002). We also examined the effects on the apical ectodermal ridge and expression of Fgf genes, including Fgf4, which

Table 2 Effects on host chick wing digit pattern of grafting DEAE soaked in various concentrations of Forskolin Concentration of Forskolin (μM)

n

0 50 100 150 250

8 6 8 7 8

Normal digit pattern 234

8 4 3 5 7

Additional 2 include (22)34, 2234.

Type of extra digits obtained Additional blip

Additional 2

1 2 1

1 2 1 1

Additional/ Transformed 3 [(33)4]

1

Polarizing activity (%)

Embryos with extra digits (%)

0 6.3 15.6 4.7 3.1

0 34 62 29 13

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Fig. 3. Expression of Shh, Fgf8, Fgf4, Ptc and Gli1 in chick limb buds 24 h after grafts of either anterior or posterior limb bud cell cultures treated for 24 h with or without Forskolin. Graft outlined. (A–C) Ventral view; Shh and Fgf8. (A′–C′) Edge on view of anterior margin, dorsal side up. Grafts of anterior control (A, A′), anterior Forskolin (B, B′), posterior Forskolin (C, C′) cultures. Note the extent and morphology of Fgf8 expression in ridge, expression stronger in panels B′ and C′, weaker in panel A′. (D–G). Frontal view; Fgf4. Grafts of anterior control (D, D′), anterior Forskolin (E, E′), posterior control (F, F′), posterior Forskolin (G, G′) cultures. (H–K) Ventral view; Ptc. Grafts of anterior control (H), anterior Forskolin (I), posterior control (J) posterior Forskolin (K) cultures. (L–O) Dorsal view; Gli1. Grafts of anterior control (L), anterior Forskolin (M), posterior control (N) posterior Forskolin (O) cultures. Note the light ectopic Gli1 expression around graft of anterior Forskolin-treated cultures (with arrows); stronger expression around grafts of posterior control and posterior Forskolin-treated cultures (with arrows).

contributes to a positive feed-back loop that co-ordinates signaling between polarizing region and ridge (Zuniga et al., 1999). Forskolin-treated grafts had a clear effect on the apical ridge (Figs. 3A′–C′). After 24 h, the ridge had grown over all the grafts, but in limb buds with grafts of Forskolin-treated cultures of anterior limb mesenchyme cells Fgf8 was expressed more widely anteriorly (Fig. 3B′) than in limb buds grafted with untreated cultures. Furthermore, the morphology of the Fgf8 expressing anterior apical ridge appeared more similar to the posterior ridge of normal limb buds and to anterior ridge Fgf8 expression following grafts of posterior limb mesenchyme cells

(compare Figs. 3B′ and C′). The Fgf4 expression domain was also shifted anteriorly and was wider in limb buds with grafts of anterior limb mesenchyme cells treated with Forskolin (Fig. 3E compare with contralateral limb E′). A shift in Fgf4 expression anteriorly was also seen in limb buds with grafts of both treated and untreated cultures of posterior limb mesenchyme cells, but the widening of the Fgf4 domain seemed more marked after Forskolin treatment (compare Figs. 3F and G; F′ and G′ are contralateral limbs). Ptc, a direct target of Hh signaling, was expressed at high levels in grafts of Forskolin-treated anterior limb mesenchyme cultures and in both treated and untreated posterior limb

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mesenchyme cultures but not in untreated anterior limb mesenchyme cultures (Figs. 3H–K). Ptc expression did not appear to be induced in host tissue adjacent to the grafts suggesting that this consequence of Forskolin treatment is cell autonomous. By contrast, another target gene, Gli1, was not expressed within any of the grafts (Figs. 3L–O) but a faint rim of expression was seen in host tissue around grafts of Forskolintreated anterior limb mesenchyme cultures and more strongly around grafts of treated and untreated posterior limb mesenchyme cell cultures (with arrows in Figs. 3M–O). Expression of genes that appear to be normally repressed in anterior regions of the limb bud by Gli3R, Hoxa13, Gremlin, dHand and Bmp2 was also modified by grafts of Forskolin treatment anterior limb mesenchyme (Fig. 4). Hoxa13 was detected in grafts of Forskolin-treated cultures of anterior limb mesenchyme cells, as well as in grafts of both treated and untreated cultures of posterior limb mesenchyme cells, but not in grafts of untreated cultures of anterior limb mesenchyme cells (Figs. 4A–D). Hoxa13 expression was not induced in

host limb buds. Expression of Gremlin, which is involved in the Shh-Fgf feedback loop, was also similarly detected in grafts of Forskolin-treated cultures of anterior limb mesenchyme cells, treated and untreated cultures of posterior limb mesenchyme cells but not in grafts of untreated cultures of anterior limb mesenchyme cells (Figs. 4E–H). However, expression was patchy in the graft and not seen in every grafted limb of Forskolin-treated anterior mesenchyme (n = 3/ 4). Marked changes were also seen in Gremlin expression in the host limb buds and expression extended towards grafts of Forskolin-treated cultures of anterior limb mesenchyme cells and both treated and untreated cultures of posterior limb mesenchyme cells (Figs. 4 F, G, H). Gremlin expression in host limb buds grafted with untreated anterior mesenchyme cells was normal. Expression of dHAND was upregulated in limbs with grafts of Forskolin-treated cultures of anterior limb mesenchyme cells (n = 1/4), treated (n = 4/4) and untreated (n = 3/4) cultures of posterior limb mesenchyme cells but not in limbs with grafts of untreated cultures of anterior limb

Fig. 4. Expression of Hoxa13, Gremlin, dHAND and Bmp2 in chick limb buds 24 h after grafts of either anterior or posterior limb bud cells cultures treated for 24 h with or without Forskolin. Graft outlined. (A–D) Ventral view; Hoxa13. Grafts of anterior control (A), anterior Forskolin (B), posterior control (C), posterior Forskolin (D). (E–H) Dorsal view; Gremlin. Grafts of anterior control (E), anterior Forskolin (F), posterior control (G), posterior Forskolin (H) cultures. Host expression extended and Gremlin is also expressed in graft (compare F–H with E). (I–L) Dorsal view; dHAND. Grafts of anterior control (I), anterior Forskolin (J), posterior control (K), posterior Forskolin (L) cultures. Ectopic expression around graft in panels J–L compared to I. (M–P) Dorsal view; Bmp2. Grafts of anterior control (M), anterior Forskolin (N), posterior control (O), posterior Forskolin (P) cultures. Ectopic expression around the graft in panels N–P compared to M.

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mesenchyme cells (n = 4/4; Figs. 4I–L). Bmp2 expression was extended and detected around the grafts of Forskolin-treated cultures of anterior limb mesenchyme cells (n = 1/4), treated (n = 4/4) and untreated (n = 3/4) cultures of posterior limb mesenchyme cells and not in limbs with grafts of untreated cultures of anterior limb mesenchyme cells (Figs. 4M–P). Altogether, these data show that downstream target genes of Shh signaling can be activated in graft and host tissue in the absence of the ligand. We also attempted to perform wholemount in situ hybridization analysis for these genes on limb buds grafted with DEAE paper soaked in Forskolin, but unfortunately, the detection solution reacted with the DEAE paper making the results impossible to interpret.

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Effects of manipulating PKA activity Forskolin elevates cAMP and should lead to an increase in PKA activity. Therefore, the effects on PKA activity of treating micromasses of limb mesenchyme cells with Forskolin were measured using incorporation of 32P in a known PKA substrate. As predicted, Forskolin treatment led to increased PKA activity (Fig. 5A). The basal PKA level was slightly higher in cultures of posterior limb mesenchyme cells than in cultures of anterior limb mesenchyme cells. After 24-h Forskolin treatment, cultures of both anterior and posterior limb mesenchyme cultures showed an increase in PKA activity compared to controls, confirming the predicted cAMP-driven rise in PKA

Fig. 5. PKA activity in cultures after Forskolin treatment and effects of altering PKA activity by retroviral infection. (A) PKA activity determined using a radioactive assay in micromasses after 24 h cultured with or without Forskolin. Forskolin treatment increases PKA activity more markedly in anterior cells. (B) RCAS-eGFP, eGFP expression in entire right wing, anterior right leg but not left wing or leg. (C, D) RCAS-aPKA, (C) Wing, digit pattern 2(33)4. (D) Leg, duplicated digit 1, pattern (11)234. Note increased cartilage, humerus, radius (C); femur (D). (E, F) RCAS-dnPKA, (E) Wing, 3 extra digits anteriorly probably all 2s, digit pattern (22)(22)34. (F) Leg, duplicated digit 1, digit pattern 11234.

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activity. The increase was more marked in anterior cultures. Furthermore, we found that this increase in PKA activity was maintained in cultures at 48 h (data not shown). We do not know, however, how long high level PKA activity would be maintained in the cells once grafted into the limb bud. cAMP has many other functions in the cell so to test whether the extra digits, that form following Forskolin treatment of anterior cultured limb bud cells, are a direct consequence of altering PKA levels, we manipulated PKA activity using RCAS viruses. To increase PKA activity, we used a virus containing an active mutant of catalytic subunit C of PKA (aPKA), and to decrease PKA activity, we used a virus containing the regulatory subunit in which cAMP binding sites have been mutated (dnPKA; Mellon et al., 1989). These constructs have previously been used in other organisms to manipulate PKA levels (Hammerschmidt et al., 1996; Ungar and Moon, 1996). We infected the presumptive chick wing bud region with these viruses by injecting concentrated virus into the coelom of stage 13–14 embryos adjacent to the region that will give rise to the right wing. Concentrated virus containing eGFP (RCAS-eGFP) was injected as a control. In the first series of experiments, we used normal White Leghorn chick embryos (wl); in the second series, pathogen-free chick embryos (spf). Fig. 5 and Table 3 show the results. Injections of control RCAS-eGFP into the presumptive limb region (wl, n = 17 and spf, n = 18) did not change digit pattern (Fig. 5B). In 18/18 pathogen-free embryos, fluorescent EGFP was detected and the whole wing was infected. Not all White Leghorn embryos were infected in every run. For example, in one run (n = 11) we could detect no fluorescence in five embryos, while limbs of 3 embryos appeared to be fully infected and 3 partly infected. In some embryos (both pathogen-free and White Leghorn), expression was also observed in the leg, mainly in digits 1 and 2. When RCAS-aPKA was injected into the presumptive limb region, 4.5% of White Leghorn embryos and 18.8% of pathogen-free embryos (wl, n = 2/44 and spf, n = 3/16) had a change in digit pattern (Figs. 5C and D). The altered digit pattern in the wing consisted of either an extra digit 2 in three cases, in one case in the wing, digit 3 was bifurcated resulting in a digit pattern 2(33)4. In one case in the leg, digit 2 was duplicated resulting in a digit pattern 12234. In limbs of some

RCAS-aPKA-injected embryos, chondrogenesis was affected and the humerus (wl; n = 4/44, spf; n = 11/16) or femur (wl; n = 0/44, spf; n = 5/16) was thicker and shorter (Fig. 5D) than those in the uninfected contralateral limbs. On some occasions, tibia and fibula were fused and the radius was missing. When RCAS-dnPKA was injected into the presumptive limb region, 23% of White Leghorn embryos and 42% of pathogenfree embryos (wl, n = 9/39 and spf, n = 8/19) had extra digits anteriorly. Sometimes limbs had more than one extra digit at the anterior margin of the limb bud (Fig. 5E); in these cases, all the extra digits looked similar but could be fused and occasionally truncated (these were scored as digit 2 for calculating polarizing activity). Following injections of both types of viruses, digit duplication was sometimes observed in the leg (Figs. 5D, F) but not the wing and in one case (for RCAS-dnPKA) both wing and leg had extra digits. For calculating polarizing activity, the duplications obtained in the leg were added to the ones obtained for the wings and the sum of all the wing patterns and leg duplications were divided by the total number of embryos. Effects of Forskolin on Gli3 protein and levels of PKA in normal limb buds Digit formation in the anterior region of the limb is normally suppressed by Gli3R therefore we monitored the effects of 24 h of Forskolin treatment on the processing of Gli3 protein in micromass cultures of chick limb cells by Western blot analysis and compared this to the distribution of the Gli3 protein (Gli3Fl and Gli3R as the antibody cannot distinguish between Gli3A and Gli3Fl) in limb tissue divided into anterior (A), middle (M) and posterior (P) regions (see Fig. 1A; n = 4 for each type of culture/tissue). Gli3Fl was present at similar levels throughout the limb bud whereas Gli3R was higher in the anterior part of the limb bud than in the posterior part (Fig. 6A). Forskolin treatment of limb mesenchyme cultures appeared to increase protein levels of both Gli3Fl and Gli3R in cultures of both anterior and posterior cells (Figs. 6B and C). Measurements of the intensity of bands in triplicate blots normalized with respect to alpha tubulin showed that the ratio of Gli3Fl/Gli3R in normal limb buds was 1.4 in the posterior half and 0.25 in the anterior half. When cells were cultured for

Table 3 Effects on digit pattern of modifying PKA activity by infecting limb buds with RCAS viruses Virus

RCAS-eGFP RCAS-aPKA RCAS-dnPKA

Type of eggs used

n

Normal spf Normal spf Normal spf

17 18 44 16 39 19

Type of extra digits obtained Normal digit pattern 234 17 18 42 13 30 11

Additional blip

2 1

Additional 2 (or in leg additional 1)

2 1 5 8

Additional 2 include (22)34, 2234, (22)(22)34. Additional 1 includes (11)234, 11234, 111234. a In both these cases an additional digit was formed, i.e., 2334 and 12234.

Additional 3 or transformed 3 (or in leg 2)

2a 2 0

Polarizing activity (%)

Embryos with extra digits (%)

0.00 0.00 1.10 7.81 9.30 11.18

0.00 0.00 4.54 18.75 23.07 42.11

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axis (Fig. 6E). PKA activity was graded across the anteroposterior axis of stage 19 limb buds being highest in the posterior region, lowest in the anterior region and intermediate in the middle. At stages 20 and 21, PKA activity was also highest at the posterior, as at stage 19, but the difference between activity in posterior and anterior regions was much less and activity was lowest in the middle (Fig. 6E). Discussion

Fig. 6. Gli3 protein levels in cell cultures and limb buds and PKA activity in normal limb buds. (A) Western blot for Gli3 in anterior, middle and posterior thirds of limb buds. (B) Western blot for Gli3 in untreated and treated cultures of anterior (AC, AF) or posterior (PC, PF) limb mesenchyme cultures and in limb buds cut into anterior, middle and posterior parts. (C) Ratio of full-length Gli3 (Gli3Fl) versus Gli3R in treated and untreated cultures of anterior or posterior limb mesenchyme and in limb buds cut into anterior and posterior parts. Increase between AC and AF is significant (p < 0.007) but not between PC and PF (p > 0.174). Error bars represent the standard deviation from the mean value of the three experiments. (D) RT-PCR for Gli3 and GAPDH in untreated and treated cultures of anterior (AC, AF) or posterior (PC, PF) limb mesenchyme. (E) PKA activity in chick anterior, middle and posterior limb buds at stages 19, 20 and 21 of development. PKA activity generally low anteriorly, high posteriorly. Error bars as in panel C.

24 h this ratio was maintained, while Forskolin treatment leads to an increase in Gli3Fl/Gli3R. This increase was significant in anterior treated cultures (p < 0.007) but not in cultures of posterior treated cultures (p < 0.174). The increase in the ratio of Gli3Fl/Gli3R in both anterior and posterior cultures is mostly due to an increase in Gli3Fl, although Gli3R also increases but to a lesser extent (Fig. 6C). This increase in total Gli3 protein (Fl and R) was not due to a change in transcription of Gli3 as semi-quantitative RT-PCR did not show any change in the level of Gli3 transcripts in anterior control versus anterior Forskolin-treated cultures and in posterior control versus posterior Forskolin-treated cultures (n = 3; Fig. 6D). Finally we determined endogenous PKA activity in different regions of normal chick limb buds across the anterior–posterior

Our main finding is that anterior wing bud cells treated with Forskolin form an extra digit 2 or digit 2 is replaced by a digit 3 (i.e., the most anterior digit of the wing is posteriorized). This appears to occur via activation of the Shh signaling pathway in the absence of ligand. Forskolin treatment leads to changes in the ratio between full-length Gli3 and Gli3R and genes downstream of Shh signaling are expressed. We show that this effect of Forskolin can be mimicked, at least in part, by increasing PKA activity directly and that the digit patterns resulting from these manipulations are similar to those obtained after Forskolin treatment. Decreasing PKA activity also induced changes in digit pattern with one or sometimes more than one extra digit 2 forming at the anterior of the wing (digit 1 in the leg). PKA activity appears to be graded across the anteroposterior axis of early chick limb buds suggesting that local levels of PKA might play a role in normal digit patterning. Altogether, our results suggest that PKA has a positive role in Shh signaling in the chick limb bud in addition to acting as a negative regulator. The changes in digit pattern and gene expression induced by Forskolin indicate that the Shh pathway has been activated in anterior mesenchyme and resemble changes in digit pattern obtained with low doses or short exposure to Shh (Yang et al., 1997). In many systems Forskolin has been reported to antagonize Shh signaling. However, there is a precedent for Forskolin activation of the vertebrate Shh pathway in left/right (L/R) patterning of the body (Kawakami and Nakanishi, 2001; Rodriguez-Esteban et al., 2001). Ectopic application of Forskolin to early chick embryos induced expression of Nodal and Pitx2, downstream targets of Shh signaling which specify L/ R asymmetry, in the absence of ligand. Furthermore, Forskolin did not appear to antagonize endogenous Shh signaling in the early chick embryo, similar to our findings in the limb. We have shown that the positive effect of Forskolin on anterior mesenchyme can be mimicked by elevating PKA levels directly. When we infected chick limbs with a virus containing an activated PKA, additional digits formed or the existing anterior digit became posteriorized, albeit in a small number of cases. In these limbs, occasionally, the cartilage of the rest of the limb was affected (Fig. 6C and D). An increase in chondrogenesis in response to Forskolin has previously has been described by Solursh et al. (1981) in micromass culture. This stimulation of Hh signaling by activated PKA contrasts with a previous finding in the CNS of zebrafish where overexpression of activated PKA suppressed cell types normally associated with Hh signaling (Hammerschmidt et al., 1996; Epstein et al., 1996; Ungar and Moon, 1996).

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In our experiments, in which we grafted Forskolin-treated cells into limb buds, there was also evidence for a nonautonomous effect. It has been known for many years that Forskolin and cAMP increase gap junctional communication between cells. In addition, there is recent direct evidence that cAMP diffusing into neighboring cells via gap junctions can mediate PKA phosphorylation in these cells (Ponsioen et al., 2006). Thus an increase in gap junctional communication coupled with intercellular diffusion of cAMP might explain the effects of grafts of Forskolin-treated cells on host cells. Interestingly, we have previously shown that gap junctional communication plays a role in digit patterning in the chick limb (Allen et al., 1990). It is not clear in our system whether other effects of Forskolin treatment, which are not directly mediated by PKA, also contribute to inducing the digit changes. For example, there is evidence that Forskolin can activate Akt/PKB (Sable et al., 1997; Filippa et al., 1999), which affects GSK3, a component of the Wnt signaling pathway (Frame and Cohen, 2001). In addition the effect of Forskolin on PKA could also be affecting other signaling pathways as demonstrated by Chen et al. (2005). We have shown that infecting chick limbs with a virus containing dnPKA also induced additional digits. With this treatment, however, unlike infection with activated PKA or treatment with Forskolin, multiple extra digits were sometimes produced. In a preliminary analysis of gene expression in limbs of embryos infected with dnPKA, a small patch of ectopic Hoxd13 expression was detected in one case, consistent with what would be expected if Gli3R was decreased. In other wings, Alx4 expression was extended (n = 1), Grm expression was reduced (n = 1) while expression of Ptc, Gli1 and Bmp2 appeared unaltered (n = 12 for each gene). Thus, although this analysis in part supports the idea that the extra digits that develop ectopically after infecting with dnPKA are due to de-repression of Gli3R, this remains to be substantiated. In Drosophila, PKA is known to exert both negative and positive effects on Hh signaling. Phosphorylation of Ci, the Drosophila homologue of Gli, by PKA is necessary for the production of Ci-75, which functions as a repressor (Jiang and Struhl, 1995; Chen et al., 1998; Price and Kalderon, 1999). The positive effect of PKA on Hh signaling is not so extensively studied but has been reported to be mediated through effects on Smo (Jia et al., 2004; Zhang et al., 2004; Apionishev et al., 2005), Ci itself (Wang and Holmgren, 2000) and activation of other, as yet unknown, factors (Zhou et al., 2006). As vertebrate Smo lacks critical PKA phosphorylation sites (reviewed Huangfu and Anderson, 2006), it seems unlikely that the activation of Shh signaling we observe after PKA treatment is mediated by effects on Smo. Furthermore, with respect to the importance of PKA phosphorylation of Ci, experiments in insects using different driver lines have given contradictory results (Chen et al., 1999; Price and Kalderon, 1999; Wang et al., 1999; Wang and Holmgren, 2000). Thus even in insects the exact targets of PKA that are required for activation of Hh signaling and that might operate in vertebrates still remain to be fully elucidated.

Other possible mechanisms by which PKA could be involved in Gli3 function in vertebrates are suggested by the effects of PKA on the functioning of other transcription factors, such as PBX1, where phosphorylation by PKA is necessary for nuclear import and functioning of this protein (Kilstrup-Nielsen et al., 2003) and dHAND, where PKA phosphorylation has been suggested to modulate dimerization properties with other factors (Firulli et al., 2003, 2005). dHAND was one of the Shh targets that we found to be ectopically expressed in host limbs with Forskolin-treated anterior mesenchyme grafts. However, it seems unlikely that PKA acts via dHAND in inducing the pattern changes that we see since hyperphosphorylation of dHAND results in limb truncation and not additional digits (Firulli et al., 2003). We found that Forskolin treatment of cultures of anterior and posterior limb mesenchyme increased the levels of both full-length Gli3 and Gli3R and that the ratio between fulllength Gli3 and Gli3R increased by the same amount in anterior and posterior mesenchyme. However, the ratio of fulllength Gli3/Gli3R in Forskolin-treated anterior cells is still lower that that found in untreated posterior mesenchyme or the posterior region of normal limb buds. This could explain why the duplications obtained are similar to those resulting from low level Shh treatment. Our data and data from others (for example Davey et al., 2006) suggest that it is not only the levels of Gli3 protein that are important but the ratio between full-length and repressor forms of the protein. The fact that we do not see a significant difference in digit pattern after grafts of treated and untreated of the posterior mesenchyme suggests that the system is saturated. In normal limbs, there is a gradient of PKA activity, with high activity posteriorly and low activity anteriorly. The high activity in the posterior part of the limb bud is surprising since Gli3R levels are lower in this region of the limb. One possibility is that high PKA may be part of a feedback mechanism to restrain the extent of polarizing activity in the posterior region of the limb bud. Acknowledgments We thank Andrew Bain for the skeletal staining, Dr. Andrea Münsterberg for the PKA viruses, Dr. Elizabeth Farrell for the RCAS-EGFP and Dr. Dave Becker for the comments on the manuscript. This work was supported by the MRC and The Royal Society. Eva Tiecke was funded by a Boehringer Ingelheim Fonds studentship and the MRC. References Allen, F., Tickle, C., Warner, A., 1990. The role of gap junctions in patterning of the chick limb bud. Development 108, 623–634. Apionishev, S., Katanayeva, N.M., Marks, S.A., Kalderon, D., Tomlinson, A., 2005. Drosophila smoothened phosphorylation sites essential for Hedgehog signal transduction. Nat. Cell Biol. 7, 86–92. Chen, Y., Gallaher, N., Goodman, R.H., Smolik, S.M., 1998. Protein kinase A directly regulates the activity and proteolysis of cubitus interruptus. Proc. Natl. Acad. Sci. U. S. A. 95, 2349–2354. Chen, C.H., von Kessler, D.P., Park, W., Wang, B., Ma, Y., Beachy, P.A., 1999. Nuclear trafficking of cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression. Cell 98, 305–316.

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