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???displayArticle.abstract??? Fibroblast growth factors (FGFs) comprise a family of signalling molecules with essential roles in early embryonic development across animal species. The role of FGFs in mesoderm formation and patterning in Xenopus has been particularly well studied. However, little is known about FGF16 in Xenopus. Using in situ hybridisation, we uncover the expression pattern of FGF16 during early Xenopus laevis development, which has not been previously described. We show that the zygotic expression of FGF16 is activated in the mesoderm of the early gastrula as a ring around the blastopore, with its first accumulation at the dorsal side of the embryo. Later, FGF16 expression is found in the otic vesicle, the branchial arches and the anteriorpituitary, as well as in the chordal neural hinge region of the tailbud. In addition, we show that FGF16 can activate the MAPK pathway and expression of sp5 and sp5l. Like FGF16, sp5 is expressed in the otic vesicle and the branchial arches, with all three of these genes being expressed in the tailbud. These data provide evidence that FGF16 is present in the early mesoderm and can activate the expression of developmentally important transcription factors.
Fig. 1. Phylogenetic analysis of predicted FGF families. FGF family groupings are represented by brackets. Bold red brackets indicate the FGF9 subfamily: FGF9, FGF16 and FGF20. Protein sequences were aligned using MUSCLE. The evolutionary history was inferred by using the Maximum Likelihood method based on the Jones-Taylor-Thornton (JTT) matrix-based model (Jones et al., 1992). The percentage of trees in which the associ- ated taxa clustered together is shown next to the branches (500 bootstrap support). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Labels are as follows: xl = Xenopus laevis; gg = Gallus gallus; hs = Homo sapiens.
Fig. 2. Expression analysis FGF16, sp5 and sp5l in gastrula stage X.laevis em- bryos. In situ hybridisation analysis was used to detect transcripts for FGF16 (A-C), sp5 (D-F), and sp5l (G-I) during gastrulation. Stages 10 posterior/vegetal (A, D, G) 11 posterior/vegetal view (B, E, H) and 12 (C, F, I) posterior view. Arrow indicates dorsal blastopore lip in all panels.
Fig. 3. Developmental expression pattern of FGF16 in X.laevis embryos. In situ hybridisation analysis was used to detect transcripts for FGF16 at several developmental stages in X. laevis embryos. At least 15 embryos were analysed at each stage (st). (A,B) St 26, lateral and anterior views respectively. (C,D) St 35, lateral and magni ed lateraltail views respectively. Abbreviations are as follows: anteriorpituitary (ap), otic vesicle (ov), plate mesoderm (pm), branchial arches (ba) and chordo- neural hinge (cnh).
Fig. 4. Developmental expression pattern of sp5 and sp5l in X.tropicalis embryos. Analysis of sp5 and sp5l expression by whole-mount in situ hybridisation of X. tropicalis embryos; at least 15 embryos were analysed at each developmental stage for each probe. (A,B) sp5 expression at neurulast18, lateral and dorsal views respectively. White triangles show faint expression in the posterior region of the embryos. (C) sp5 expression at tailbud st25, lateral view. (D,E) sp5l expression at neurulast18, lateral
and dorsal views respectively. (F) Transverse section of the embryo in (E) at the level of the dashed line. (G) sp5l expression at tailbud st26, lateral view. (H) Transverse section of embryo in (G) at the level of the dashed line. (I-K) sp5 expression at tailbud st31, lateral view. (L,M) sp5l expression at tailbud st31, lateral view. Abbreviations: Midbrain (MB), Neural crest (NC), Forebrain (FB), Otic placode (OP), MidbrainHindbrain Boundary (MHB), Migrating crest cells (MCC) Neural fold (NF), Neural tube (NT), Branchial Arches (BA), Otic Vesicle (OV).
Fig. 5. Hydrophobicity plots for FGF9 family. Kyte and Doolittle hydrophobicity plots for (A) FGF4 and FGF9 sub-family members, (B) FGF9, (C) FGF16, and (D) FGF20. Scores above 0 (represented by the dotted line) indicate hydrophobic amino acids; sites of high hydro- phobicity are depicted as shaded areas. FGF4 has a “classic” signal sequence (SS) for co-translational secretion, but the members of the FGF9 subfamily have an internal hydrophobicity region which is as- sociated with secretion.
Fig. 6. FGF16 activates the MAPK pathway and activates sp5 and sp5l expression (A) Western blot to detect dpERK in uninjected control animal caps compared and animal caps injected with 50 pg of mRNA coding for FGF16 mRNA. Antibodies to diphospho-ERK (dp-ERK) and total ERK were incubated on the same blot sequentially after stripping. (B) Control animal caps after 3 days culture. (C) Animal caps expressing FGF16 from sibling embryos to those shown in (B). (D) RT-PCR on cDNA derived from whole embryos at stage 12 and on animal cap explants cultured to stage 12 that were either uninjected control explants or explants expressing FGF4 or FGF16. Expression of known FGF targets, xbra (27 cycles), egr1 (27 cycles) and cdx4 (27 cycles), as well as sp5 (25 cycles), sp5l and rpl8 loading control. Water, in which H2O replaced cDNA, and - RT, where no reverse transcriptase was used, functioned as negative controls.
