Development, Growth DifferentiationVolume 56, Issue 8 p. 555-572 Original Article Free Access Effect of FGFR inhibitors on chicken limb development Dana Horakova, Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech RepublicSearch for more papers by this authorPetra Cela, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno, Czech RepublicSearch for more papers by this authorPavel Krejci, Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech RepublicSearch for more papers by this authorLukas Balek, Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech RepublicSearch for more papers by this authorSimona Moravcova Balkova, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Clinic of Stomatology, St. Anne\'s Faculty Hospital and Faculty of Medicine, Masaryk University, Brno, Czech RepublicSearch for more papers by this authorEva Matalova, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Department of Physiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech RepublicSearch for more papers by this authorMarcela Buchtova, Corresponding Author Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Author to whom all correspondence should be addressed. Email: buchtovam@vfu.czSearch for more papers by this author Dana Horakova, Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech RepublicSearch for more papers by this authorPetra Cela, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno, Czech RepublicSearch for more papers by this authorPavel Krejci, Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech RepublicSearch for more papers by this authorLukas Balek, Department of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech RepublicSearch for more papers by this authorSimona Moravcova Balkova, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Clinic of Stomatology, St. Anne\'s Faculty Hospital and Faculty of Medicine, Masaryk University, Brno, Czech RepublicSearch for more papers by this authorEva Matalova, Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Department of Physiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech RepublicSearch for more papers by this authorMarcela Buchtova, Corresponding Author Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic Institute of Animal Physiology and Genetics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic Author to whom all correspondence should be addressed. Email: buchtovam@vfu.czSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Fibroblast growth factor (FGF) signalling appears essential for the regulation of limb development, but a full complexity of this regulation remains unclear. Here, we addressed the effect of three different chemical inhibitors of FGF receptor tyrosine kinases (FGFR) on growth and patterning of the chicken wings. The inhibitor PD173074 caused shorter and thinner wing when using lower concentration. Microinjection of higher PD173074 concentrations (25 and 50mmol/L) into the wing bud at stage 20 resulted in the development of small wing rudiment or the total absence of the wing. Skeletal analysis revealed the absence of the radius but not ulna, deformation of metacarpal bones and/or a reduction of digits. Treatment with PD161570 resembled the effects of PD173074. NF449 induced shortening and deformation of the developing wing with reduced autopodium. These malformed embryos mostly died at the stage HH25–29. PD173074 reduced chondrogenesis also in the limb micromass cultures together with early inhibition of cartilaginous nodule formation, evidenced by lack of sulphated proteoglycan and peanut agglutinin expression. The effect of FGFR inhibition on limb development observed here was unlikely mediated by excessive cell death as none of the inhibitors caused massive apoptosis at low concentrations. More probably, FGFR inhibition decreased both the proliferation and adhesion of mesenchymal chondroprogenitors. We conclude that FGFR signalling contributes to the regulation of the anterior-posterior patterning of zeugopod during chicken limb development. Introduction Cartilage and bone formation is preceded by mesenchymal cell condensation, arising either via the accumulation of cells towards a prospective ossification centre or by a failure of cells to move out of this centre (Langille 1994a,b). The size and position of the condensed area must be precisely controlled and its formation is critical to guide the proper cell differentiation during development of the skeletal element (Moftah etal. 2002). At the cellular level, the condensation results from epithelial-mesenchymal interactions that govern cell proliferation and direction of migration in neighbouring cell populations. The fibroblast growth factor (FGF) signalling system regulates many developmental processes including skeletogenesis, brain patterning, branching morphogenesis or limb development (Mariani etal. 2008). The FGF ligands control cell proliferation and differentiation by activating four trans-membrane tyrosine kinase receptors, the fibroblast growth factor receptors (FGFR1-4) (Mohammadi etal. 1997). In mammals, there are 22 known FGF ligands with at least five of them being expressed in the embryonic limb and seven during craniofacial development (Martin 1998; Nie etal. 2006; Hatch 2010). FGF10 initiates limb formation from the lateral plate mesoderm (Ohuchi etal. 1997) and induces formation of the apical ectodermal ridge (AER), which is a major signalling centre in the limbs (Saunders 1948; Bell etal. 1962; Fernandez-Teran & Ros 2008). Then, FGFs produced by AER contribute to the maintenance of proliferation in the limb mesenchyme, resulting in proximal-distal growth and patterning of limbs (Niswander etal. 1993; Tickle 2002). Later, FGF signalling is involved in chondrogenesis where it contributes to the regulation of cell survival and chondrocyte differentiation (Ornitz 2005). Three FGFRs are expressed during limb development and have been shown to be essential for skeletogenesis (De Luca & Baron 1999): Fgfr1 is expressed in the developing limb mesenchyme, Fgfr2 in mesenchymal condensations, and Fgfr3 in developed growth plate cartilage (Peters etal. 1992, 1993). Several small chemicals were produced to inhibit the kinase activity of FGFRs. These compounds exhibit different activities towards individual FGF receptors, as well as the off-target activities against other receptor tyrosine kinases (Hamby etal. 1997; Batley etal. 1998; Krejci etal. 2010). Notably, most developmental studies still preferentially use SU5402 (Szabo-Rogers etal. 2008; Sato etal. 2011; Bouffant etal. 2012; Jacques etal. 2012), although this compound inhibits FGFRs with relatively low efficiency and it is active against other FGFR-unrelated tyrosine kinase receptors (Sun etal. 1999). To overcome some of these disadvantages, we used three novel chemical FGFR inhibitors, NF449, PD161570 and PD173074, to address the role of FGF signalling in the regulation of chicken limb development. Both PD161570 and PD173074 inhibit FGFR activation via interference with ATP binding in contrast to NF449, which targets FGFR kinase activity independently of ATP (Mohammadi etal. 1998; Skaper etal. 2000; Krejci etal. 2010). PD161570 is a selective FGFR inhibitor at lower concentrations (IC50=40nmol/L), while inhibiting also platelet derived growth factor (PDGFR) and epidermal growth factor receptor (EGFR) at higher concentration. PD173074 is a selective FGFR3 and FGFR1 inhibitor (IC50=5, 21.5nmol/L) at lower concentrations while targeting also VEGFR at very high concentrations. NF449 inhibits FGFR3 (IC50=0.2–0.5μmol/L) (Krejci etal. 2010); however it targets also the P2X and P2Y receptors (Braun etal. 2001; Gonzalez etal. 2005; Rettinger etal. 2005). All three compounds were recently demonstrated to target FGFR signalling during regulation of several developmental processes both in vivo and in vitro, including gastrulation, olfactory system development, primitive endoderm development, prostate bud and neural cell proliferation (PD173074), primary osteoblast differentiation (PD161570) and chondrocyte proliferation (NF449) (Stevens etal. 2003; Kuslak & Marker 2007; Krejci etal. 2010; Oki etal. 2010; Jia etal. 2011; Yang etal. 2011; Gibson etal. 2012). In this study, we address the effects of NF449, PD161570 and PD173074 on chicken wing development. As all of these compounds exhibit different activity profiles towards FGFRs, the comparison of their actions allows the examination of limb development under quantitatively and qualitatively different FGFR mediated stimuli. We demonstrate that a complete inhibition of FGFR signalling suppresses limb bud outgrowth leading to absent limbs. More importantly, a partial inhibition of FGFR signalling reveals a previously unknown role of FGFR signalling in skeletogenesis. We show that a lower threshold of the FGF stimulus is necessary for induction of mesenchymal condensations within the posterior field of the growing wing bud in contrast to the anterior field where low levels of FGFR inhibition completely eliminate the cartilage formation, resulting in the loss of corresponding zeugopod skeletal elements. Fertilized chicken eggs (ISA brown) were obtained from Integra farm (Zabcice, Czech Republic). Eggs were incubated in a humidified forced air incubator at 37.8°C, and embryos were staged according to Hamburger and Hamilton (1951). All procedures were conducted following a protocol approved by the Laboratory Animal Science Committee of the University of Veterinary and Pharmaceutical Sciences (Brno, Czech Republic). Application of FGFR inhibitors, skeletal analysis, histological processing and whole-mount in situ hybridisation NF449, PD161570, PD173074 and SU5402 were purchased from Tocris Biosciences (Bristol, UK). Inhibitors were applied into the chicken right wing bud at the stage HH20-22. Micromanipulator (Leica, Germany) and a microinjector (Eppendorf, Germany) were used to better target the selected area of application. Three injection sites (anterior, posterior, distal) were used to inhibit FGF signalling in the whole limb bud equally (Fig.1H,I). PD161570 and PD173074 were applied at concentrations of 1, 25, 50mmol/L, and NF449 was used at concentrations of 0.2, 0.5 and 1.0mmol/L. Inhibitors were diluted in dimethylsulfoxide (DMSO) and treatment with DMSO was used as a negative control. SU5402 was soaked into AG1X2 beads for 1h at room temperature followed by wash in MEM. We could not use beads for both PD inhibitors and NF449 as these compounds precipitated on beads when using higher concentrations. Figure 1Open in figure viewerPowerPoint Skeletal analysis of wings after the application of PD173074. At the lower concentration (1mmol/L), there was a partial reduction (A′) or full absence of the radius (B′). Moreover, digitus alulae was shorter (B′). All distal limb structures were absent in embryos with a missing humerus (C′) or those with a stump of the humerus (D′). Coracoid was reduced in size (C′,D′) and scapula was smaller when 25mmol/L concentration of the inhibitor was applied. (E,F) The highest concentration of the inhibitor (50mmol/L) led to the absence of the humerus (E′,F′) and also skeletal structures more distal to the humerus were missing. The scapula was shorter and thinner (E′) or absent (F′). The coracoid was shorter and thinner (E′) or completely missing (F′). The clavicle was shorter and deformed (E′); only in one case, it was absent (F′). The left (control) wing was also affected at this high concentration as the radius was rudimental (E) or missing (F), with shorter metacarpus and missing fingers (F). (A–F) Left control wings versus right experimental wings (A′–F′) are shown. (G,G′) DMSO injected control embryos with normal skeletal phenotype (H,I) Sites of injections into the right wing bud were visualized by Trypan Blue. s – scapula, c – coracoid, cl – clavicle, h – humerus, r – radius, u – ulna, m – metacarpus, mj – digitus major, mi – digitus minor, a – digitus alulae. Following the inhibitor application, embryos were collected after 10–12days of incubation, fixed in 100% ethanol, stained with Alizarin red/Alcian blue solution, and cleared in KOH/Glycerol, as previously described (Plant etal. 2000). For histology, both right (treated) and left (control) wings of selected stages between HH25 to HH29 were collected and fixed in 4% paraformaldehyde overnight at 4 °C. Whole-mount in situ hybridization was performed as described previously (Song etal. 2004) with using digoxygenin (DIG) labelled Fgf8 probe (Dubrulle & Pourquie 2004). Embryos were injected with BrdU 2h before their collection. Limbs were fixed in 4% paraformaldehyde overnight at 4 °C. After deparaffinization and rehydration, the sections were pretreated in 2N HCl and in 0.1% trypsin/phosphate-buffered saline (PBS), in the humidified chamber (37 °C) for 30 and 10min, respectively. Non-specific secondary antibody binding was inhibited by incubation in a blocking serum for 20min at room temperature (RT). The slides were incubated with the primary monoclonal antibody (BrdU, 1:30, B8434, Sigma-Aldrich, Czech Republic) for 1h at RT. For cell adhesion detection, antigen retrieval was performed for 5min in citrate buffer in the water bath (97 °C). A primary N-cadherin antibody (1:50, ab1203, Abcam, Cambridge, UK) was incubated for 1h at RT in the humidified chamber. The biotinylated secondary antibody and the Vectastain streptavidin-FITC complex (1:200, SA-5001, Vector Laboratories, Peterborough, UK) conjugations were performed for 30min at RT. Sections were counterstained by DAPI. Negative control was obtained by omitting the primary antibody from the labelling protocol. For apoptosis, the nuclear DNA fragmentation was labelled in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, according to manufacturer\'s protocol (ApopTag Peroxidase in situ Apoptosis Detection Kit; Chemicon, Temecula, CA, USA). Counterstaining with haematoxylin was performed. A negative control was obtained by omitting the enzyme from the labelling protocol. Mesenchymal cultures were established from the anterior and posterior parts of forelimb buds of stage HH20 chicken embryos. Wing buds were dissected into Puck′s saline A (PSA, 0.8% NaCl, 0.04% KCl, 0.035% NaHCO3, 0.1% glucose), digested by Dispase II (1U/mL, Sigma, Czech Republic) and filtered to obtain a single cell population. Cells were re-suspended at a density of 2×107 cells/mL of medium and plated in 10μL aliquots in BD Biocoat fibronectin-coated, 35mm culture dishes (BD Biosciences, Heidelberg, Germany). Cells were left to adhere for 1h in the incubator before 2mL of culture media (F12/Dulbecco\'s modified eagle medium [DMEM] supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, glutamine, 1% ascorbic acid and 10mmol/L β-glycerol phosphate) containing PD173074 or PD161570 was added to each well. Treatment with DMSO was used as a negative control. For Alcian blue staining, micromass cultures were placed in 4% paraformaldehyde, washed in PBS/0.1mol/L HCl and stained with 0.5% Alcian blue overnight. Images were taken with a Leica S6D (Leica Microsystems, Germany) microscope, and pixels of blue colour were counted using Adobe Photoshop 7.0 software (Adobe Systems Incorporated, San Jose, CA, USA). All of the results are shown as means±SD of five independent biological replicates. Each experiment was repeated three times. For peanut agglutinin (PNA) detection, micromass cultures were collected after 3days of incubation, fixed in 4% paraformaldehyde and processed through methanol series in PBS containing 0.1% Tween 20. Samples were then washed three times, blocked for 30min and incubated with the primary antibody PNA conjugated to Rhodamine (1:200, RL-1072, Vector Laboratories, Peterborough, UK). Following the primary antibody incubation, sections were washed in PBS and covered by ProLongGold anti-fade medium (Invitrogen, Prague, CR); individual cultures were photographed in dark field. Haematoxylin was applied for background staining to visualize small cartilage nodules. To assess cell viability, micromass cultures were collected after 3 and 6days of incubation. Cells were counted before plating and at the time of collection and equal amounts of cells were incubated with 200nmol/L tetramethylrhodamine ethyl ester (Invitrogen, Prague, CR) for 15min at 37 °C. Viability measurements were acquired on flow cytometer (Accuri C6; BD Biosciences, San Jose, CA, USA). Cells were lysed with 1% sodium dodecyl sulfate (SDS)lysis buffer. Protein concentrations were measured with BioRad DC Protein Assay Kit II (500-0112, Bio-Rad) and equalized. β-mercaptoethanol and bromophenol blue were added to samples before boiling. Proteins were transferred to an Immobilon-P membrane (IPVH00010, Millipore) and determined using phospho-p44/42 and p44/42 antibodies (cs4376 and cs9102, Cell Signaling Technology, Boston, MA, USA). Signal was visualized using SuperSignal West Femto Chemiluminescent Substrate (34095, Thermo Fisher Scientific Inc., Rockford, IL, USA). For analysis of Dusp6 expression, forelimb buds of stage 20 chicken embryos were isolated. Micromass cultures were made separately from the anterior and posterior parts of wings. In selected cultures, medium was supplemented with 50ng/mL of FGF2 (FGF-basic, R&D Systems, Minneapolis, MN, USA) or with 50nmol/L PD173074 or with both PD173074 and FGF2. Micromass cultures were lysed with RLT Lysis buffer (Mini RNeasy Kit; Qiagen, Germany) and samples were collected at 8 or 24h after the treatment. Total RNA was isolated from tissues using the Mini RNeasy Kit (Qiagen, Germany), following the manufacturer\'s instructions. Complementary DNA (cDNA) was synthesized using SuperScript VILO (Invitrogen, Darmstadt, Germany). Real-time polymerase chain reaction (PCR) reactions were performed using the primers and MGB probes (TaqMan) designed by the company (Applied Biosystems/Ambion, Austin, TX, USA) on Realplex4 Eppendorf (Germany). The comparative CT method was used for analysis as described in User Bulletin #2 (Applied Biosystems/Ambion, Austin, TX, USA). Cycle conditions were as follows: 95°C – 10min, 40×cycles of 95°C – 15s and 62.5°C – 1min. The following primers and Taqman probes were used: Hprt1: Gg03338900_m1, Dusp6: Gg03364898_m1 (Applied Biosystems TX, USA). To uncover the most prominent impacts of FGFR inhibitors PD173074, PD161570 and NF449 on the wing external phenotype and embryonic survival, we tested their effect in three different concentrations. As chicken wing development is initiated at the stage HH17 of the embryonic period, we injected inhibitors into stages HH20–22 when the wing anlagen had already protruded as the bud but the mesenchymal cells are not specified as pre-cartilaginous cells. Low concentration of PD173074 (1mmol/L; n=68; Fig. S1A′,B′; Table1) caused the deformation and shortening of the right wing after treatment in most cases (33.8%). Furthermore, we observed rudimental limbs (7.4%) or limb stumps (11.8%). At a higher concentration (25mmol/L; n=47; Fig. S1C′,D′; Table1) all embryos exhibited an abnormal external phenotype where the most common phenotype was the absence of the right wing (51.1%), rudimental wing (21.3%) or wing stump (21.3%). Occasionally, cleft lip (6.4%) or shortening of mandible (6.4%) occurred (Fig. S2A–F). The highest used concentration (50mmol/L; n=19, Table1; Fig. S1E′,F′) affected also all collected embryos. The most common phenotype was the absence of the right wing (84.2%); only a few wing stumps were observed (15.8%). The left wing was affected in three embryos (15.8%), possibly due to the inhibitor distribution via blood circulation. Cleft lip occurred in five embryos (26.3%), and a shorter mandible was seen in one case (5.3%) (Fig. S2G–I). Morphological changes of embryos treated with PD161570 were similar to those induced by PD173074. At low concentration (1mmol/L; n=34, data not shown, Table S1A), PD161570 caused thinner and shorter wing (44.1%) or the embryos had a wing rudiment that remained in the wing bud stage (29.4%). When a higher concentration (25mmol/L) was applied, all embryos exhibited altered phenotype (n=30; Table S1A) where the phenotypes ranged from the total reduction of the wing (36.7%) to a thinner and shorter limb (33.3%). Occasionally, cleft lip (3.3%), a short mandible (3.3%) or a deviated beak was observed (6.7%). Some changes in phenotype were observed also on the contralateral left side without treatment (6.7%). The highest concentration (50mmol/L, n=32; Table S1A) often led to the absence of the right wing (37.5%) or rudimental wing (46.9%) with occasional presence of shorter mandibles (6.3%). The application of NF449 led to higher mortality of embryos. Therefore, we also analyzed embryos, which died around the stage HH25, when only the external morphology could be evaluated. Here, we provide information separately for those embryos that were collected early, and for those surviving into the late developmental stage (HH34) when skeletal analysis was possible (Table S1B). Following the application of the lowest concentration (0.2mmol/L; n=11; Table S1B, data not shown), we observed shorter and thinner wings, smaller autopodium or wing stumps. Both higher concentrations of NF449 (0.5mmol/L; n=18 and 1mmol/L; n=26) caused a high ratio of abnormal embryos among early-collected embryos, while surviving embryos exhibited milder phenotype (Table S1B). With increasing concentration of the inhibitor, we observed higher embryonic mortality. Low concentration of SU5402 (1mmol/L; n=72 and 1.7mmol/L; n=15; Table S1C) caused only mild phenotype where right wing was shorter only in a few embryos (4% for 1mmol/L, 13% for 1.7mmol/L) but the number of affected embryos increased up to 25% when using even higher concentration (2.5mmol/L; n=28). Following the application of the highest concentration (3.3mmol/L; n=23), we observed shorter and thinner wings (69.6%) or wing rudiments (13%). As external morphology was largely affected by FGFR inhibitor treatment, we also analyzed skeletal changes underlying the external phenotype of embryos that survived 10–12days after inhibitor injection. All wing and girdle skeletal elements were stained with Alizarin red/Alcian blue solution to uncover possible different effects on wing segments in the proximo-distal as well as anterior-posterior axis. At the 1mmol/L concentration (n=11; Table S2A, Fig.1A′,B′), PD173074 caused a partial reduction or full absence of the radius (27.3%) on the right (injected) side of wing; the left (control) side of the skeleton was intact. Furthermore, the metacarpus was deformed and fingers were either shorter or missing (18.2%). At higher concentration (25mmol/L, n=27; Table S2A) PD173074 led to the reduction of ribs on the right side in 33.3% of the collected embryos (Fig.1D′). All distal limb structures were absent in embryos with a missing humerus (59.3%, Fig.1C′) or those with a stump of humerus (37%, Fig.1D′). If the humerus was shorter, the radius and digitus alulae were missing and the ulna, metacarpus, digiti minor and major were present but shorter. Other changes included a thinner and shorter coracoid (44.4%, Fig.1C′) or its absence (29.6%). In addition, there was a phenotype on the left untreated wing (14.8%) where mostly the radius and digiti alulae were missing. The application of PD173074 at the highest concentration (50mmol/L; n=13; Table S2A) resulted in absence of the humerus (84.6%, Fig.1E′,F′) or its reduction while structures more distal to the humerus were missing. The scapula was typically shorter and thinner (23.1%, Fig.1E′) or absent (38.5%, Fig.1F′). The coracoid was normal in only one embryo; in most embryos it was missing (46.2%) or shorter and thinner (46.2%). The clavicle was mostly shorter and deformed (53.8%, Fig.1E′). The left (non-injected) wing was more affected at this high concentration in comparison to lower concentrations. In most cases (38.5%), left radius was missing, with shorter or missing metacarpus and fingers, the antebrachium and autopodium were missing (23.1%) or radius was reduced (15.4%). All control embryos injected with DMSO showed normal skeletal morphology (Fig.1G′). In most embryos treated with the lowest PD161570 concentration (1mmol/L; n=10; Table S2B), we observed a complete loss of the radius (70%; Fig. S3A′) or reduced radius with thinner and shorter appearance (30%, Fig. S3B′) accompanied by deformation of the autopodium (Fig. S3B′). The humerus was often affected (50%), while the scapula or ribs exhibited normal morphology. Higher concentrations (25mmol/L; n=18; Table S2B) led to a shorter or thinner scapula (33.3%, Fig. S3C′,D′). The humerus was frequently affected (77.8%); however, the most commonly affected structure was the radius and digitus alulae (100%). In most cases, the defect involved the radius only, whereas the ulna was normal. In other cases, the ulna was missing when the humerus was absent or developed as a stump. Ribs were occasionally affected, being absent or shorter (11.1%, Fig. S3D′). At 50mmol/L (n=19; Table S2AB), PD161570 caused skeletal changes of the coracoid, which was mostly thinner and shorter (52.6%) and occasionally missing (15.8%, Fig. S3F′). The clavicle was shorter (31.6%) and the scapula thinner and shorter (63.2%, Fig. S3E′). When the humerus was absent (47.4%, Fig. S3F′) or developed as a stump (36.8%, Fig. S3E′), then all distal structures were also absent. The left wing was affected in only one embryo (5.3%), with deformation of the scapula and digiti alulae. The application of the lowest concentration of NF449 (0.2mmol/L; n=4; Table S2C; Fig. S4A′,B′) produced normal skeleton formation in all of the examined embryos. A higher concentration of NF449 (0.5mmol/L; n=11; Table S2C) affected mostly segments of the fingers (36.4%), and the metacarpus was thinner (27.3%). Furthermore, the radius (18.2%) or ulna was thinner (18.2%) (Fig. S4C′,D′). The microinjection of the highest concentration of NF449 (1mmol/L; n=11; Table S2C) caused shortening of the radius along with the ulna (18.2%; Fig. S4E′). Moreover, the metacarpus was shorter along with shorter or absent fingers Fig. S4E′,F′). The application of the low concentrations of SU5402 (1mmol/L; n=72; and 1.7mmol/L; n=15; Table S2D; Fig. S5 A′, B′) caused only minor effect on proximal bones, where the humerus was shorter and thinner (13%). A higher concentration of SU5402 (2.5mmol/L; n=28; Table S2D) also affected mostly the humerus, which was reduced in size (25%) or absent (4%). Only mild phenotype was observed on the radius, ulna and metacarpus (4%). The highest concentration (3.3mmol/L; n=7) led to a complete loss of the humerus (29%) or its shortening (57%). When the humerus was affected then the radius, ulna as well as all distal structures were absent (43%; Fig. S5D′) or reduced in size (43%, resp. 29% for metacarpus, Table S2D). The phenotype of hindlimbs resembles the wing phenotype with a more severe effect on the anterior skeleton As we demonstrated anterior-posterior differences in response to FGFR inhibition in wing zeugopod elements, we next asked whether the same phenotype can be achieved also in the hindlimbs. There are morphological differences in chicken zeugopod elements – while the anterior bone of wing (radius) is smaller, the anterior skeletal element of hindlimb (tibia) is larger in contrast to posterior elements. We injected PD173074 into hindlimb buds at HH stages 20–22. As NF449 produced only mild phenotypes and both PD173074 and PD161570 exhibited similar phenotypes, PD173074 was selected for the following analyses. We used only a lower PD173074 concentration (1mmol/L) to see possible variations in phenotype without full inhibition of cartilage formation. Anterior skeleton (tibia) was more severely affected than posterior bone (fibula) similarly to wings, where nine tibias (52.9%; n=17) exhibited high level of malformed phenotype but only three fibulae (17.6%; n=17; Fig.2). Figure 2Open in figure viewerPowerPoint Skeletal analysis of hindlimbs after the application of PD173074. (A–C) The treatment of hindlimbs by low inhibitor concentration (1mmol/L) led to shortening of tibia while fibulae were affected only slightly (E–G). (D,H) Differences between right and left (control-untreated) skeletal elements were measured and plotted to the graph showing the frequency of observations. Further, we used the wing bud micromass approach to address possible mechanisms underlying the inhibition of chondrogenesis. Only PD173074 was used in these experiments, since it gave consistent morphological phenotype in wings (Fig.1). Alcian blue staining was applied to evaluate the onset of chondrogenesis in differentiating micromass cultures (Fig.3A–F). Previous studies using stage HH24-26 wings showed high variability in patterns of chondrogenesis according to different media composition (Downie & Newman 1994, 1995). Therefore, we first analyzed the pattern of normal chondrogenesis in micromass cultures. As our microinjections were situated in the period between stages HH20 and HH22, we established high-density cultures from chicken wing at this early bud developmental stage. In addition, the skeletal analysis showed differences in responsiveness to the treatment in the anterior and posterior areas of the wing. To analyse possible differences at the cellular level, we cut the wing buds into anterior and posterior parts at the time of collection and established micromass cultures from both areas separately (Fig.3G). The basal level of chondrogenesis was different in the micromass cultures prepared from the anterior and posterior parts of wing buds with less of proteoglycan production in the anterior parts (Fig.3A,B). Figure 3Open in figure viewerPowerPoint PD173074-mediated inhibition of extracellular matrix production in micromass cultures derived from the early wing mesenchyme. Micromass cultures were established from the anterior and the posterior wing bud mesenchyme at the stages between HH20 and HH22. Alcian blue staining was applied after 6days of incubation to evaluate chondrogenesis. (A,B) A lower production of the extracellular matrix was observed in the cultures established from the anterior parts of wing bud. (C,D) The treatment with lower concentration (10nmol/L) of PD173074 led to small inhibition of chondrogenesis, while higher concentrations (100nmol/L) of the FGFR inhibitor significantly decreased the chondrogenesis (E,F). (G) Graphical designation of cutting edge limiting the anterior and the posterior wing areas used for cultures. (H) The number of blue pixels was counted for each treatment and statistical significance was analyzed by ANOVA. All results are showed as means±SD of five independent biological replicates. , medium; , 10nmol/L; , 100nmol/L Treatment with PD173074 demonstrated the inhibition of chondrogenesis in comparison to control cultures (Fig.3C–F). A statistically significant decrease of chondrogenesis was observed at the 100nmol/L PD173074 concentration compared to 10nmol/L, which caused no significant change in comparison to control micromasses (Fig.3H). Chondrogenesis and production of the cartilaginous extracellular matrix were completely inhibited in micromasses treated with 1μmol/L of PD173074 (data not shown). We observed almost complete inhibition of chondrogenesis in anterior cultures at the concentration of 100nmol/L, where only a few cartilage nodules were formed (Fig.3E). To visualize the influence of PD173074 on early mesenchymal condensations, we used the staining for peanut agglutinin (PNA), which is a galactose-specific lectin. PNA recognises B-D-gal-Nac-D-gal, a terminal carbohydrate moiety of cell surface glycoproteins and preferentially binds to cells in condensations (Davies etal. 1990; Gotz etal. 1991; Milaire 1991a,b; Oakley etal. 1994). Micromass cultures established from early wing buds were treated with two different concentrations of PD173074 (10 and 100nmol/L) as described above (Fig.4A–F). Cultures were fixed after 3days of incubation. The treatment with PD173074 decreased PNA expression in a dose-dependent manner, with significant inhibition observed at the concentration of 100nmol/L PD173074 (Fig.4E′,F′). Moreover, the cultures prepared from the posterior wing area exhibited a higher degree of PNA staining in contrast to the anterior cultures. Figure 4Open in figure viewerPowerPoint Early inhibition of cartilage nodule formation after PD173074 treatment. Peanut agglutinin (PNA) labelled by rhodamine (red) was used to visualize early mesenchymal condensations. Micromass cultures were established from the anterior (A,C,E) and the posterior (B,D,F) areas of wing bud. Cultures were collected and stained after 3days of incubation. There was a higher level of PNA expression in the posterior (B′) cultures compared to the anterior ones (A′). The treatment with PD173074 (C′,E′) decreased PNA expression in a dose-dependent manner in comparison to medium only (A′). Cultures prepared from the posterior limb area exhibited similar nodule inhibition after PD173074 treatment (D′, F′) with significant inhibition observed at the concentration of 100nmol/L (E′,F′). The background of micromasses was stained by Haematoxylin to visualize small nodule formation (blue). To better visualize a formation of small nodules, we stained the background of micromasses by Haematoxylin. The number of cell clusters was reduced in the anterior micromasses after 3days of incubation (compare Fig.4A,B), confirming the difference between both areas during the early formation of mesenchymal condensations. The inhibitory influence of PD173074 was also clearly visible with regard to density and the number of clusters formed (Fig.4E,F; compared to 4A,B). Both, PNA and Haematoxylin stainings demonstrated the early inhibition of chondrogenesis by PD173074 during formation of cell clusters formation. To identify cellular processes underlying the effect of PD173074 on chondrogenesis, we first determined the inhibitor effect on cell apoptosis and proliferation. Embryos were microinjected with PD173074 and collected during stages when chondrogenesis proceeds in the wing mesenchyme (Fig.5). First, we tested the effect of PD173074 (1mmol/L) on apoptosis activation to analyze whether disruption in the antebrachial bone development might be a consequence of an increased apoptosis in mesenchymal cells. Only samples with an external phenotype were selected for this analysis. By stage 27, the antebrachium already exhibited formation of two cartilages for the radius and the ulna with positive Alcian blue staining (Fig.5M). In contrast, the treated right wing showed only limited cartilage formation (Fig.5N). There was obvious single mesenchymal condensation after the application of 1mmol/L PD173074 (Fig.5N). These wing anlagens were able to produce only one posterior bone in the zeugopod area. Even more apparent differences between the treated and control sides were observed at later developmental stages (Fig.5O,P,Q,R). Treatment with PD173074 did not increase the number of apoptotic cells at these later stages (Fig.5M′–R′). TUNEL-positive cells were localized only in the areas of physiological cell death (Montero etal. 2001) during cartilage development (Fig.5M–R). We also analyzed earlier stages of limb bud and found that the number of TUNEL-positive cells was increased around injection area 3h after treatment, while they mostly disappeared 6h after treatment. As we used DMSO for inhibitor dilution, we treated limb buds with only DMSO as a control and we also observed an increase in the number of apoptotic cells around injection area 3h after treatment (Fig. 5C,F). However, the process was compensated in 6h time point collected samples (Fig.5I,L) therefore this temporal increase of apoptosis does not seem to influence the final phenotype (Fig.1G′). Figure 5Open in figure viewerPowerPoint Localization of apoptotic cells after the treatment with PD173074. (A,A′,D,D′,G,G′,J, J′) Left control (non-injected) wing exhibited only few apoptotic cells at early stage of limb bud. (B,B′,E,E′) PD173074 injected right wing as well as (C,C′,F, F′) DMSO treated wing contained increased number of apoptotic cells around injected areas 3h after injection. The number of apoptotic cells decreased at 6h after injection in both PD173074 (H,H′,K, K′) or DMSO treated wings (I,I′,L,L′). (M,O,Q,) At later stages of development, left control wing exhibited formation of two cartilages, one for the radius and one for the ulna labelled by positive Alcian blue staining. (N,P,R) PD173074 (1mmol/L) treated right wing showed only limited cartilage formation with only one mesenchymal condensation. These wing anlagens were able to produce only one posterior bone in the zeugopod area. Furthermore, the shape of the autopodium was altered. Differences between control and treated wings became even more apparent at later developmental stages (compare O,P and Q,R). TUNEL-positive cells (arrows) were localized only in the areas of physiological cell death (M′,M′′,O′,O′′,Q′, Q′′). The number of apoptotic cells was not increased at these later stages after FGFR inhibitor treatment (N′,N′′,P′,P′′,R′,R′′). TUNEL-positive cells were visualized by DAB (brown cells) and negative cells by Hematoxylin (blue cells). Scale bar=100μm. We further tested the effect of high PD173074 concentrations on embryos. While embryos survived the application of 25 and 50mmol/L concentrations of the inhibitor, the number of apoptotic cells increased in the treated wing (data not shown). This suggests that the total reduction of the wing and all bones including the girdle was underlined by a non-specific increase of apoptosis in the wing bud; therefore, these concentrations should not be used for in vivo studies. Similarly, when haematoma was observed in wings during collection, apoptosis was increased in the middle of bud already 6h after treatment (data not shown). Based on previous in vitro as well as in vivo observations, apoptosis does not seem to be the main mechanism involved in the inhibition of cartilage formation in the antebrachium. To test if FGFR inhibition influenced cell proliferation, we applied PD173074 (1mmol/L) to the wing bud and performed BrdU analysis on alternative slices. We found only a slight decrease of BrdU-positive cells in the distal area of the wing (Fig.6). Proliferating cells were dispersed throughout the fore limb mesenchyme and, after their differentiation into chondrocytes, the number of BrdU-positive cells was equally decreased in both treated as well as control fore limbs (data not shown). Figure 6Open in figure viewerPowerPoint Decrease of proliferation followed PD173074 treatment. (A,A′) BrdU-positive cells were dispersed through the control untreated wing except of the central area, where cartilaginous blastema (c) started to differentiate. (B,B′) The number of proliferating cells was decreased in the posterior region; however, there was not any obvious downregulation of proliferation in the central area. All exhibited specimens were collected 24h after treatment. (C) The number of proliferating cells were counted also in micromass cultures established separately from the anterior and the posterior parts of the wing bud. Decrease of proliferation was observed in each conditions, however, statistically significant differences were found only after the application of higher concentration of the inhibitor in both anterior and posterior areas after 3days of incubation and in the posterior area after 6days of incubation. All results are showed as means±SD of three independent biological replicates. The total number of cells in control cultures with medium was established as 100%. BrdU-positive cells were labelled by FITC (green cells) and nuclei by DAPI (blue cells). Scale bar=100μm. , medium; , PD 10nmol/L; , PD 100nmol/L Furthermore, we analyzed changes in cell numbers within the micromass cultures (Fig.6C). Cells were counted 3 or 6days following the treatment with PD173074, and cell numbers were determined by counting the whole cell population. The total number of anterior cells increased by about 2.5 times after 3days of incubation and 5.5 times after 6days in medium when compared to the start-up number of cells (data not shown). There was a smaller increase after 10nmol/L treatment – about two times after 3days of incubation and five times after 6days. The total number of cells was even less elevated after 100nmol/L treatment – 1.5 times after 3days and 4.5 times after 6days of incubation (data not shown). Therefore, cell proliferation was decreased by about one-third after FGFR inhibitor treatment (Fig.6C). We did not observe any significant differences in cell proliferation between micromass cultures isolated from the anterior versus the posterior wing bud; the trend of inhibition was similar (Fig.6C). We thus conclude that the inhibition of cell proliferation contributes to PD173074-mediated loss of chondrogenesis in the wing bud mesenchyme; however, this cannot fully explain the skeletal phenotype and differences between anterior and posterior areas. The apical ectodermal ridge (AER) plays a critical role in regulation of proliferation of the adjacent limb mesenchyme; AER removal leads to reduced proliferation and/or increased cell death in the limb mesenchyme, resulting in a total loss of limb (Dudley etal. 2002; Sun etal. 2002). The external phenotype in our treated embryos collected at early developmental stages exhibited smaller and thinner autopodium when using low concentrations of FGFR inhibitors and a total wing loss when using higher concentrations, thus resembling the AER removal experiments. Based on this evidence, we proposed that morphology and signalling of AER could be modified after the FGFR inhibitor treatment. We used Fgf8 as a marker of the apical ectodermal ridge (Lewandoski etal. 2000). In agreement with our hypothesis, we observed downregulation of Fgf8 after PD173074 treatment (1mmol/L) at later time points (Fig.7). The level of downregulation was consistent with the level of phenotype from absent expression to small decreases of expression (Fig.7). Figure 7Open in figure viewerPowerPoint Fgf8 expression in the wing anlagen after PD173074 treatment. (B,B′,D,D′) There was not obvious difference in Fgf8 expression intensity at 3h time point after treatment or (F, F′,H,H′) 6h after PD173074 treatment (1mmol/L). (J,L,N) Down-regulation of Fgf8 was observed 24h after FGFR inhibitor treatment. The intensity of down-regulation was consistent with the level of phenotype from absent expression (J′) to small decreases of expression (L′,N). Left untreated side is shown for the comparison (A,C,E,G,I,K) including left wing details (A′,C′,E′,G′,I′,K′,M). The formation of cellular condensation is a critical step in chondrogenesis. We analyzed peanut agglutinin (PNA) expression as an early marker of cartilage formation. We next observed PNA expression in the middle of limb anlagens in the area of pre-cartilaginous cartilage in the control left wing (Fig.8A′,B′). In older stages, two positive areas of differentiating cartilage were detected. Treatment with PD173074 (1mmol/L) resulted in the disruption of PNA expression with many positive clusters of cells or individual positive cells out of areas of differentiating cartilage (Fig.8C′,D′). Figure 8Open in figure viewerPowerPoint PNA and N-cadherin expressions were altered after PD173074 treatment. (A′,B′) PNA expression was located in the middle of limb anlagens in the area of pre-cartilaginous mesenchyme in the control left wing. (C′,D′) The expression was disrupted after the treatment with PD173074 (1mmol/L), where small positive clusters of cells surrounded the areas of differentiating cartilage. (E′,F′) N-cadherin expression was low in the middle area of cartilage formation in the control wing, while the expression was strong around differentiating cells particularly at the leading edge of chondrogenic blastema. (G′,H′) The pattern of N-cadherin expression was altered in the wings treated with PD173074 (1mmol/L) with high expression around proximal end of forming cartilage and moreover around cartilage in separated positive clusters. PNA-positive regions were labelled by Rhodamine (red), N-cadherin-positive areas by FITC (green cells) and negative cells by DAPI (blue cells). (A,B,E,F) Left untreated side labelled by DAPI is shown for the comparison to right PD173074 treated side (C,D,G,H). Scale bar=100μm. Furthermore, adhesion of cells to each other as well as to the extracellular matrix controls chondrocyte differentiation. N-cadherin contributes to adherent junctions and is expressed in cellular condensations during limb development, while it is absent in differentiated cartilage later (Oberlender & Tuan 1994a). We observed the disruption of N-cadherin expression after PD173074 (1mmol/L) treatment (Fig.8G′,H′). At the earlier developmental stage, we observed high expression in the cartilaginous core of the left control limb in contrast to the weak expression in the treated limb (data not shown). At the later stage (HH29), there was downregulation of expression in the middle area of the control wing, while the expression was strong around differentiating cells (Fig.8E′,F′). Wings treated with PD173074 also exhibited down-regulation in the area of cartilage formation, which was a much smaller size compared to the control wing; furthermore, expression of N-cadherin around the core was high, disrupted and separated cell clusters were observed (Fig.8H′). Our labelling of early cartilage formation showed that the disruption of zeugopod development after FGFR inhibitor treatment arose at premature stages when mesenchymal condensation was initiated and the disruption of cell adhesion plays a role in the mesenchymal patterning of the zeugopod. To further explore possible mechanism of PD173074 inhibition and its effect on FGF signalling, we analyzed the activity of pERK in micromass cultures established from anterior and posterior parts of limb buds. We observed inactivation of pERK in the both cultures following the treatment with PD173074 (Fig.9A). On the other hand, the ectopic activation of FGF pathway by FGF2 treatment led to immediate induction of pERK. Figure 9Open in figure viewerPowerPoint Downstream targets of FGF signaling were affected by PD173074 treatment. (A) The activity of pERK in micromass cultures established from the anterior and posterior parts of fore limb buds was rapidly activated by FGF2 and inactivated by PD173074 treatment as analyzed by western blot. (B,C) Dusp6 expression was increased in cultures treated by FGF2 and downregulated by PD173074 at 8h as well as 24h after the treatment. , anterior; , posterior. Moreover, FGFR signalling is necessary for Dusp6 transcription (Li etal. 2007), which is expressed during chicken wing development in similar expression pattern to FGFR2. While at stage 22–23 Dusp6 is uniformly expressed in distal limb mesenchyme, later in development it is shifted to the anterior domain of chicken limb bud (Uejima etal. 2010). We analyzed the changes in Dusp6 expression in micromass cultures treated with PD173074 or FGF2 as a control of pathway activation. We observed the increase of Dusp6 expression following cultures with FGF2 treatment (Fig.9B,C). While FGFR inhibition by PD173074 led to downregulation of Dusp6 expression in both analyzed time points (8h, 24h); however, differences were not statistically significant at 8h time point. Here, we provide a detailed analysis of the effect of three chemical FGFR inhibitors on the chicken limb development. The application of different concentrations allowed us to finely modulate the levels of FGF signalling in developing wings and to reveal differences in anterior-posterior patterning effects. In line with the basic pharmacological profiles provided by the producer, we observed different effects of the applied inhibitors on chicken embryos where the most striking phenotype was observed after the application of PD161570; even a small concentration (1mmol/L) affected about 76% of the treated embryos. The smallest effect was evident after the application of NF449, where about half of the embryos exhibited some affected phenotype. While embryos treated with PD161570 and PD173074 survived even very high concentrations with severe phenotypes in the experimental wing and mild phenotypes in other parts of the body, respectively, the application of NF449 led to high lethality of chicken embryos. The highest concentration (1mmol/L) that could be applied to achieve a reasonable survival rate was much lower in contrast to the 50 times higher amount in the case of the other tested inhibitors. We expect that this lethality was caused by the effect of NF449 on other receptors than FGFR3. Previously, it was shown that NF449 also inhibits P2X receptors, and also P2Y receptors at higher concentrations (Braun etal. 2001; Gonzalez etal. 2005; Rettinger etal. 2005). The intravenous application of NF449 into mice led to prolonged bleeding, and inhibition of the platelet aggregation (Hechler etal. 2005). We expect that delayed coagulation after microinjection of the inhibitor into the wing bud may cause the higher lethality of our embryos. We show that slight modulation of FGFR activity in developing avian limb bud affect chondrogenic pattern formation with the strongest effect on the anterior part of the limb. The phenotype increased with rising concentrations of FGFR inhibitors and corresponded to variability in different mouse combined mutants (Mariani etal. 2008). The chicken stylopod is formed only by the humerus. If the stylopod was affected, then we observed severe malformations of the zeugopod and autopod structures. From all chicken wing segments, the zeugopod and autopod were influenced by the FGFR inhibitor treatment most commonly. The chicken zeugopod is formed by the radius and ulna where the ulna is located posteriorly and is much larger than the anterior radius. The FGFR inhibitor treatment exhibited an asymmetrical influence on zeugopod bones as the anterior radius was reduced first. Whereas, in mice, there are no obvious size differences between the zeugopod bones; however, a similar asymmetrical reduction of the radius was observed also in the Fgf8;Fgf9 double knockout (Mariani etal. 2008). This indicates that the size of bones or cell number forming the initial mesenchymal condensation is not decisive, but the anterior cells are more sensitive to even low levels of FGF signalling inhibition. The autopod of chickens is composed of digitus alulae, which is situated on the anterior side, digitus major and digitus minor. Digitus alulae was found to be affected slightly more often than the other two fingers. A similar reduction in finger numbers as well as variability in their number was also observed in the Fgf8;Fgf9 double knockout mice (Mariani etal. 2008). A more severe phenotype was observed in the Fgf8;Fgf4 double knockout mice, as the forelimb lacked many skeletal elements including the radius and most of the digits (Mariani etal. 2008). Therefore, the phenotype in both of these knockouts demonstrated defects in anterior structure formation in a similar way as shown here after the application of synthetic inhibitors at low concentrations. Furthermore, histological analysis of treated chicken wings showed the absence of Alcian blue staining during early stages of chondrogenesis in the area of prospective radius formation. Similarly, the Fgf8;Fgf9 double knockout exhibits the absence of Sox9 expression in the anterior region of limb anlagen (Mariani etal. 2008). Higher concentrations of FGFR inhibitors led to the presence of the stylopod (humerus) while the autopod and zeugopod were absent; the humerus was often smaller and thinner. In the Fgf8−/−Fgf4−/−Fgf9+/−mice, stylopod size was also reduced along with zeugopod absence. In contrast to our experiment, they also observed small rudimental fingers in several cases (Mariani etal. 2008). We expect that a similar phenotype might be seen if the FGFR inhibitor PD173074 was used in concentration between 1 and 25mmol/L, which we did not test in the present study. The range of skeletal phenotypes observed after FGFR inhibitor treatment reflects differences in the local ability of wing mesenchymal cells to respond to FGF signals. The application of chemical FGFR inhibitor (PD173074) causes significant changes in early chondrogenesis during limb development in vivo, which was also confirmed in vitro in the micromass cultures. It was previously shown that the formation of condensation is important for initiation of cell differentiation (Hall & Miyake 1995). The treatment of micromass cultures with FGFR inhibitors demonstrated decreased labelling by PNA, which confirmed the inhibition of chondrogenesis at an early stage of condensation formation. There are three possible mechanisms that can underlie inhibition of chondrogenesis: increased apoptosis, decreased proliferation and misregulation of cell adhesions. FGFR inhibitor did not increase the number of apoptotic cells in the right wing when compared to control at the concentration of 1mmol/L, while morphological changes in zeugopod and autopod structures were obvious. Apoptosis was observed only in the anterior and posterior necrotic zones as it is physiological for the early stages of chicken limb development during their shaping (Montero etal. 2001). Therefore, apoptosis did not seem to contribute to the phenotype. Mesenchymal condensation precedes the cartilage formation. We found that the cell adhesions visualized by N-cadherin immunostaining were disrupted at the early stage of chondrogenesis after FGFR inhibitor treatment. Sparsely scattered expression of N-cadherin together with the disturbance of PNA expression confirmed that the development was disrupted at the early pre-cartilage stage. Similar findings were observed when the blocking antibody to N-cadherin, NCD-2, was used (Oberlender & Tuan 1994a,b). Cellular condensation and subsequent chondrogenesis were inhibited in vivo as well as in vitro. Interestingly, the application of NCD-2 into the chicken limb bud also led to the disruption of digit development and the asymmetric loss of zeugopod skeletal elements, where the fibula was missing in the hindlimbs and occasionally the radius in the forelimbs (Oberlender & Tuan 1994a,b). There is a high variability in the arrangement of zeugopod bones among species. In chickens and alligators, the ulna is much larger than the radius. This is in contrast to many mammalian species, where the radius is the main load-bearing bone of the forelimb and the ulna can be significantly reduced in size (e.g. in the cow) or even only rudimental and fused to the radius (e.g. in the horse). Here, we showed that the anterior cells are more sensitive to FGFR inhibitors. The application of FGFR inhibitors can help in the future to solve the puzzle of limb bone reduction during evolution. From the evolutionary point of view, it is interesting that a subtle modulation of a strength of FGF signal leads to a reduction of one of the antebrachial bones. Changes of FGF signaling could therefore account for different levels of antebrachial bones reduction during vertebrate evolution. This study was supported by the Grant Agency of the Czech Republic (304/09/0725 and 14-31540S to MB lab, P305/11/0752 to PK lab), Ministry of Education, Youth and Sports of the Czech Republic (KONTAKT LH12004) (PK), Grant Agency of Masaryk University (0071-2013) (PK), and institutional support (RVO:67985904) (MB). dgd12156-sup-0001-FigS1.tifimage/tif, 4.4 MB Fig. S1. External wing phenotype after the application of PD173074. dgd12156-sup-0002-FigS2.tifimage/tif, 7.8 MB Fig. S2. External and skeletal phenotype of the head after application of PD173074. dgd12156-sup-0003-FigS3.tifimage/tif, 2.4 MB Fig. S3. Skeletal analysis of wings after the application of PD161570. dgd12156-sup-0004-FigS4.tifimage/tif, 1.5 MB Fig. S4. Skeletal analysis of wings after the application of NF449. dgd12156-sup-0005-FigS5.tifimage/tif, 4.6 MB Fig. S5. Skeletal analysis of wings after the application of SU5402. dgd12156-sup-0006-TableS1A-B-C.docWord document, 42 KB Table S1A. External analysis of wing morphology after PD161570 treatment.Table S1B. External analysis of wing morphology after NF449 treatment.Table S1C: External analysis of wing morphology after SU5402 treatment. dgd12156-sup-0007-TableS2A-B-C-D.docWord document, 70 KB Table S2A. Skeletal analysis after PD173074 treatment.Table S2B. Skeletal analysis after PD161570 treatment.Table S2C. Summary of skeletal analysis after NF449 treatment.Table S2D. Skeletal analysis after SU5402 treatment. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Batley, B. L., Doherty, A. M., Hamby, J. M., Lu, G. H., Keller, P., Dahring, T. K, Hwang, O., Crickard, K. & Panek, R. L. 1998. Inhibition of FGF-1 receptor tyrosine kinase activity by PD 161570, a new protein-tyrosine kinase inhibitor. Life Sci. 62, 143– 150. 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