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Role of FGFs in the control of programmed cell death during limb development

¹ Departamento de Ciencias Morfológicas y Biología Celular y Animal, Universidad de Extremadura, Badajoz 06071, Spain
² Instituto Gulbenkian de Ciência, Rue da Quinta Grande, 6 Oeiras, Portugal
³ Department of Anatomy and Physiology, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK
⁴ Unidad de Investigación, Hospital Marques de Valdecilla, Santander 39008, Spain
⁵ Departamento de Anatomía y Biología Celular, Facultad de Medicina, Universidad de Cantabria, Santander 39011, Spain
⁶ Instituto Cajal, CSIC, Dr Arce 37, Madrid 28002, Spain

View larger version (95K): [in a new window]   Fig. 1. Inhibition of interdigital apoptosis by treatment with SU5402. (A,B) Left side, control (A) and right side (B) treated chick leg autopodes vital stained with Neutral Red 48 hours after implantation of a SU5402-bead (arrow). (C,D) Interdigital apoptosis 12 hours after application of a BMP-bead (*) in a normal limb interdigit (C) and in an interdigit 24 hours after implantation of a SU5402-bead (arrow; D). Note the reduced apoptotic effect of BMPs in limbs previously treated with the FGF inhibitor. (E,F) Scanning electron micrographs showing the presence of soft tissue syndactyly 4 days after interdigital application of SU5402 (F); E is the left control autopod of the same embryo. (G-P) Micromass cultures of stage 25 progress zone mesoderm showing chondrogenesis by Alcian Blue staining after 5 days of incubation (G,I,K,M,O) or by in situ hybridization for the type II collagen gene expression after 4 days of incubation (H,J,L,N,P). Control untreated cultures (G,H) and cultures treated with Noggin (100 ng/ml; I,J); SU5402 (800 ng/ml; an FGF inhibitor; K,L); SU5402 and BMP7 (800 and 50 ng/ml respectively; M,N); and BMP7 (50 ng/ml; O,P). Note that chondrogenesis is intensely inhibited by Noggin (I,J versus G,H) but not by SU5402 (K,L versus G,H) and that addition of SU5402 does not inhibit the chondrogenic effect of BMP7 (M-N vs O-P). (Q,R) Digit morphology 3 days after implantation of only a SU5402-bead at the tip of digit 3 (Q) and after combined treatment of SU5402 and BMP7 (R). Note that in both cases digit outgrowth is blocked by the inhibition of FGFs, but the growth promoting effect of BMP7 on the cartilage is not blocked giving rise to a dramatically enlarged phalanx (R).

View larger version (129K): [in a new window]   Fig. 2. Expression of FGFs and FGF receptors in the developing autopod during the stages of interdigital cell death. (A) Expression of Fgf8 in the AER at stage 31. At this stage interdigital apoptosis is present in the second and third interdigits. (B) Expression of Fgf12 at stage 29. This member of the FGF family is expressed in the progress zone mesoderm and in the interdigital regions showing a predominant distribution in the posterior part of the autopod. (C,D) Expression of FGFR1 at stages 30 (C) and 29 (D). This receptor is predominantly expressed in the surface of the condensing mesenchyme of the digital rays and in the developing tendons, but a low level of expression is also present in the undifferentiated mesenchyme. (E-G) Expression of FGFR2 in the developing autopod. (E) Whole-mount in situ hybridization after short digestion with proteinase K (20 minutes) in a stage 30 autopod showing the ectodermal expression of FGFR2 in the ectoderm. (F) Tranverse and (G) longitudinal sections of the autopod at stage 31. Note the intense expression of this receptor in the interdigital mesoderm. (H-M) Expression and regulation of FGFR3 in the developing autopod. (H) Longitudinal and (I) transverse sections of the autopod at stage 30 showing the intense expression of this receptor in the diferentiating cartilage and in the interdigital regions. (J-M) Whole-mount in situ hybridization showing the regulation of FGFR3 after interdigital application of BMP7 (K) and FGF2 (L,M). (J) Control autopod at stage 30. (K) Experimental autopod 6 hours after implantation of a BMP-bead. Note the intense up-regulation of this receptor in the treated interdigit. (L,M) Experimental autopodes 10 hours (L) and 20 hours (M) after implantation of an FGF-bead (arrow). Note that FGFR3 is initially down-regulated (L) and then up-regulated (M) by the application of FGFs.

View larger version (100K): [in a new window]   Fig. 3. Effects of FGF2 on interdigital cell death. (A,B) Interdigital cell death (vital stained with Neutral Red) in left control (A) and right treated (B) chick autopods 14 hours after implantation of a FGF-bead into the third interdigit (arrow). Note that at this time period of treatment cell death is fully inhibited by FGFs. (C) Left control and (D) right treated chick autopodes 48 hours after implantation of an FGF-bead (arrow) into the third interdigit. Note the intense increase of cell death at this time period after the application of FGF2. (E) Control and (F) experimental duck interdigital webs showing the increase in apoptosis 50 hours after implantation of a FGF-bead (*; F). (G) TUNEL labeling of interdigital cell death 30 hours after the implantation of an FGF-bead (*). (H) BrdU incorporation into the interdigital mesoderm 30 hours after the implantation of a FGF-bead. Note that cell proliferation is inhibited in the zone of induced apoptosis (compare with G). (I,J) Illustrate the inhibition of FGF-mediated cell death by co-administration of a Noggin-bead. (I) Cell death 30 hours after the implantation of an FGF-bead (arrow) and (J) its inhibition when a Noggin-bead (arrow) is co-implanted with a FGF-bead (arrowhead).

View larger version (107K): [in a new window]   Fig. 4. (A-C) Effect of FGF-beads (*) implanted into the third interdigit at stage 28 on the expression of BMP2 (A) BMP4 (B) and BMP7 (C). (D-F) Effects of beads (*) soaked in SU5402 implanted into the third interdigit at stage 28; (D,F) The expression of BMP4 and BMP7, 36 hours after the treatment; (E) expression of BMP7, 20 hours after the treatment. Note that BMPs continue to be expressed at high levels in the treated interdigits.

View larger version (92K): [in a new window]   Fig. 5. Regulation of the interdigital expression of MSX2 in chick and duck limbs by FGFs and BMPs. (A) Control chick at stage 30. (B) Upregulation of MSX2 24 hours after the implantation of a FGF-bead (*). (C) Control duck at day 9.5 of incubation. Note that MSX2 expression in the duck is restricted to the most distal region of the interdigital webs. (D) Upregulation of MSX2 in the duck 14 hours after interdigital implantation of a FGF-bead (*). (E) Down-regulation of MSX2 in the chick 30 hours after implantation of a bead incubated in SU5402. (F) Down-regulation of MSX2 in the chick 14 hours after implantation of a Noggin-bead (*). (G) Expression of MSX2 in the chick 14 hours after implantation of a FGF-bead (white asterisk) and a Noggin-bead (black asterisk). Note the up-regulation by FGFs is inhibited in the zone of influence of the Noggin-bead. (H) Expression of MSX2 in the chick 8 hours after implantation of a BMP-bead (white asterisk) 24 hours after implantation of a SU5402-bead (black asterisk). Note that in these conditions BMPs fail to upregulate MSX2.

View larger version (106K): [in a new window]   Fig. 6. (A-D) Expression of Snail in the embryonic chick limb at stages 25 (A), 26 (B), 29 (C), and 31 (D). Note the progressive increased labeling of the interdigital regions at stage 31 (D) in correlation with the establishment of INZs. (E) Expression of Snail in the duck leg autopod at day 9.5 of incubation. Note that the interdigital domains of Snail become restricted distally coincidently with the zones of interdigital cell death in the duck (compare with Fig. 3E). (F) Upregulation of Snail in the third interdigit of the duck leg 14 hours after implantation of a FGF-bead (*). (G) Duck leg bud 14 hours after implantation of a FGF-bead (white asterisk) and a Noggin-bead (black asterisk). Note that the positive effect of FGFs on Snail expression is inhibited in the zone of influence of the Noggin-bead. (H) Upregulation of Snail, 6 hours after implantation of a BMP-bead (*) into the third interdigit of the chick. (I) Down-regulation of Snail expression 6 hours after implantation of a Noggin-bead (*) into the third interdigit of a stage 28 chick embryo. (J) Upregulation of Snail in the chick 10 hours after interdigital implantation of a FGF-bead (*) at stage 28. Note that the upregulation of Snail parallels that observed for BMPs (see Fig. 4B). (K) Down-regulation of Snail, 24 hours after interdigital implantation of a bead (*) incubated in SU5402. (L) Expression of Snail in the chick interdigit 8 hours after implantation of a BMP-bead (white asterisk) 24 hours after implantation of a SU5402 bead (black asterisk). Note that in these conditions BMPs are unable to upregulate Snail expression (compare with H and K).

View larger version (78K): [in a new window]   Fig. 7. (A-C) Skeletal alterations after treatments with FGFs in specimens stained with Alcian Green showing the formation of extra cartilaginous elements after implantation of FGF-beads at stage 20 in the anterior margin mesoderm (A), posterior margin mesoderm (B) and in the progress zone (C). Arrows indicate the position of the induced extra cartilaginous elements. (D-H) Changes in the pattern of cell death after treatments with FGFs in specimens vital stained with neutral red. (D-F) Control (D) and experimental wing buds showing the inhibition of apoptosis in the PNZ (E) and in the ANZ (F) 12 hours after the implantation of FGF-beads (*). (G,H) Control and experimental limbs showing the normal pattern of ANZ (G) and the increased apoptosis 24 hours after implantation of an FGF-bead (*, H) in the anterior margin mesoderm. (I) Limb bud vital stained for cell death 24 hours after implantation in the anterior margin mesoderm of an FGF-bead (*) and a Noggin-bead (arrow). Note the intense inhibition of apoptosis in the zone of influence of the Noggin-bead (compare with H).

View larger version (137K): [in a new window]   Fig. 8. (A) Transverse section of the limb bud processed for TUNEL showing the increased cell death 24 hours after the implantation of an FGF-bead into the anterior margin mesoderm. Note that apoptosis is induced at some distance form the bead (*). (B,C) Upregulation of Snail (B) and MSX2 (C) 16 and 24 hours, respectively, after implantation of an FGF-bead (*) into the anterior margin mesoderm of the chick wing bud. (D-H) Regulation of BMP gene expression between 5 and 20 hours after implantation of FGFs in the anterior (E-G) or posterior margins (D,F,H) of the bud at stages 20-22: BMP2 (D); BMP4 (E,F); and BMP7 (G,H). Note the expanded domains of bmps in the treated limbs in comparison with the contralateral control limb. (I) Scanning electron micrograph showing the flattening of the AER 24 hours after the implantation of an FGF-bead in the anterior margin of the wing bud. Arrows indicate the anterior end of the AER.