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sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish

¹ Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch cedex, CU de Strasbourg, France
² Max Planck Institute for Cell Biology and Genetics, Dresden. c/o Department of Neurobiology University of Heidelberg Im Neuenheimer Feld 364 D-69120 Heidelberg, Germany

View larger version (65K): [in a new window]   Fig. 1. Phylogenetic tree and sequence alignment of Spry proteins. The complete peptide sequences of human (H) Spry1, Spry2 and Spry3, as well as mouse (M) and zebrafish (Z) Spry4 are aligned with the cysteine rich domain of Drosophila (D) Spry. The cysteine-rich domain is shaded and two additional short stretches of similar amino acids are boxed. Dashes indicate identical or similar amino acids, dots indicate gap that have been introduced to optimize the alignment. Stars highlight cysteine residues that are conserved among all Spry proteins. The complete protein sequences of human Spry1 and human Spry3 were deduced from genomic sequences (gb/AC026402 and gb/AC025226, respectively). The zSpry4 sequence has been submitted to GenBank Accession Number, AF371368.

View larger version (88K): [in a new window]   Fig. 2. fgf8, fgf3 and spry4 are co-expressed during the first 48 hours of embryonic development. (A-H) Blastula and gastrula stages, dorsal towards the right. (A,B) Sphere stage. Expression of fgf8 and spry4 at the dorsal margin. (C,D) Expression of fgf3 and spry4 in the marginal blastoderm of late blastula stage embryos. (E,F) Vegetal pole view of the dorsoventral expression gradient of fgf8 and spry4 at the margin of mid-gastrula stage embryos. (G,H) At late gastrula stages fgf3 and spry4 are co-expressed in the ventral forebrain primordium (arrowhead). (I-N) Segmentation stages. Anterior is upwards in (I,J) and towards the left in (K-N). (I-L) Early segmentation stages. fgf8, fgf3 and spry4 are expressed in the telencephalon (te). (I,J) fgf8 and spry4 display similar expressions in the isthmus, the hindbrain, the somites and the tail bud (tb). (K) fgf3 expression is more restricted in the hindbrain and the tail bud. (M,N) 16-somite stage. Expression of fgf8 and spry4 in the telencephalon, the midbrain-hindbrain boundary (mhb), somites and tail bud. fgf8 but not spry4 is expressed in the anterior unsegmented paraxial mesoderm (upm). spry4 but not fgf8 is expressed in the otic vesicle (ov). (O,P,S,T) 24 hour stage: anterior is towards the left. (O,P) Lateral view. fgf8 and spry4 are expressed in the telencephalon, the epiphysis (ep), the optic stalk (os) and the MHB. (S,T) Dorsal view. Both genes are expressed in the anterior otic vesicle. Posteriorly, fgf8 is detectable in the vesicular epithelium, while spry4 is expressed in adjacent cells outside of the vesicle. (Q,R) 36 hour stage, lateral view, anterior towards the left. fgf3 and spry4 are co-expressed in the branchial arches (ba), the MHB and the otic vesicle. (U-X) 48 hour stage. (U,V) Lateral view, anterior towards the left. fgf8 is expressed in the adenohypophysis, spry4 in the adjacent neurohypophysis. (W,X) Dorsal views of dissected pectoral fins. fgf8 is expressed in the apical ectodermal ridge, spry4 in the underlying mesenchyme.

View larger version (112K): [in a new window]   Fig. 3. Expression of spry4 in wild-type and acerebellar (ace) embryos. (A,C,E) Wild-type siblings. (B,D,F) Homozygous mutant embryos. (A,B) Dorsal views, anterior towards the top. (C,D) Dorsal views, anterior towards the left. (E,F) Lateral views, anterior towards the left.

View larger version (59K): [in a new window]   Fig. 4. Inhibition of Fgf3 by antisense morpholino induces loss of spry4 expression. All views are lateral, anterior towards the left, except E,F where anterior is upwards. (A,B) 30 hour stage. Injection of an anti-fgf8 (@fgf8) morpholino induces a loss of the cerebellum, similar to that observed in fgf8/ace mutant embryos. (C,D) 36 hour stage. Injection of an anti-nacre (@nac) morpholino induces complete loss of neural crest-derived pigmentation. (E,F) Late gastrula stage. Injection of an anti-fgf3 (@fgf3) morpholino induces a loss of spry4 expression in the ventral forebrain primordium (arrowhead). (G-J) Telencephalic region of five somite stage embryos. Inactivation of Fgf8 through the ace mutation (H) or @fgf8 injection (I) leads to a partial reduction of spry4 expression. (J) Complete loss of spry4 transcripts is observed after the simultaneous inhibition of Fgf8 and Fgf3 through co-injection of the corresponding morpholinos. (K,L) Pharmacological inhibition of FGF signaling by treatment with SU5402 leads to complete loss of Spry4. wt, wild type.

View larger version (92K): [in a new window]   Fig. 5. Fgf8 and Fgf3 induce spry4. (A-D) Lateral view of gastrula stage embryos stained for spry4. Dorsal is towards the right. (A) Wild type. (B,C) Widespread overexpression of Fgf3 or Fgf8 induces spry4 throughout the embryo. (D) Microinjection of fgf8 mRNA in one central blastomere of a 16-cell stage embryo induces spry4 at the animal pole, far from its endogenous expression domain. (E,F) Implantation of Fgf8-secreting beads induces spry4 expression. Lateral view (E) and dorsal close-up, anterior towards the left.

View larger version (79K): [in a new window]   Fig. 6. Effects of gain of Spry4 function on embryonic development. (A,B) Lateral cephalic views of 36 hour embryos. Overexpression of Spry4 by microinjection of 200 pg spry4 mRNA (B) induces a loss of the cerebellum and reduction of the otic vesicle (arrowhead), similar to the phenotype of ace mutant embryos (A). (C-F) Lateral view of 10-somite stage embryos stained for engrailed 2 (C,D) and for pax2.1 in (E,F). (C,E) Wild-type embryo (D,F) and embryo injected with 250pg spry4 mRNA (D,F). Expression territory of engrailed 2 (D) and pax2.1 (F) (delimited by arrowheads) appears narrower. (G,H) Dorsal view of bud-stage embryos stained for Spry4. Overexpression of Spry4 by microinjection of 200 pg spry4 mRNA (comprising the ORF only) induces loss of endogenous spry4 expression at the midbrain-hindbrain boundary (H). (I-L) Injection of 1 ng spry4 mRNA induces a range of ventralized phenotypes. 30-hour stage, anterior towards the left. Spry4 induces expansion of ventral hematopoietic derivatives (J, arrow compared with wild-type in I), a reduction of cephalic territories and a strong expansion of hematopoietic territory (K) labeled with CB588, a blood-specific marker (L). (M,N) Lateral view of late blastula stage embryo. Expression of BMP2b is expanded dorsally in embryos injected with 200 pg of spry4 mRNA (N) compared with wild-type (M). Arrowheads delimit the dorsalmost BMP2b expression in ventral non marginal blastomeres. (O,P) Lateral view of a late blastula stage embryo. Expression of an epidermal-specific gene, CG1061, is upregulated in an embryo injected with 200 pg spry4 mRNA (P). Compare with wild type (O).

View larger version (76K): [in a new window]   Fig. 7. Effect of loss of Spry4 function on embryonic development. (A,B) Dorsal view of 10-somite stage embryos stained for pax2.1, myod and shh. Inhibition of Spry4 function by injection of spry4 morpholino (@S4) induces a lateral expansion of the somites (B). (C,D) embryos at late blastula stage on lateral view. Upon injection of @spry4 expression of BMP2b is strongly reduced in ventral blastomeres. (E, F) Lateral view of embryos at the 10-somite stage, labeled for krox20, engrailed 2 (eng2) and emx1. Injection of @spry4 causes a strong enlargement of emx1 expression in telencephalon. (G,H). Lateral cephalic view of 36 hour embryos. Embryos display facial outgrowings (arrowhead) following the injection of @Spry4, probably resulting from early enlargement of telencephalon.

View larger version (37K): [in a new window]   Fig. 8 Spry4 antagonizes FGF signaling pathway in vivo. (A1) fgf8 (or fgf3) mRNA is injected with rhodamine dextran into one blastomere of the 16 cell stage embryo. (A2) If the progeny of the injected cells comes to lie in the ventral aspect of the early gastrula, a secondary embryonic axis is formed (A3). (B) At the 16-cell stage, two batches of embryos that have been previously saturated with lacZ- or spry4-mRNA are co-injected into one blastomere with eGFP- and fgf8 (or fgf3) mRNA. The GFP allows to identify ventrally located clones. Spry4 inhibits the effect of ventral Fgf8 misexpression (C) or ventral Fgf3 misexpression (D). (E) Injection of 60 pg CA-FGFR1 (CA-R1) strongly dorsalizes embryos (1) co-injection of 125 pg (2) or 250 pg spry4 mRNA (3) progressively rescues the dorsalized phenotype to wild type. (F) Localized injection of CA-FGFR1 in one blastomere at the 16-cell stage induces a strong activation of ERK at blastula stage. (G) Overexpression of spry4 inhibits activation of MAPK at gastrula stage.