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First published online 21 January 2009
doi: 10.1242/dev.027904

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FGF ligands in Drosophila have distinct activities required to support cell migration and differentiation

California Institute of Technology, Division of Biology MC114-96, 1200 East California Boulevard, Pasadena, CA 91125, USA.

View larger version (98K): [in this window] [in a new window]   Fig. 1. Dynamic expression patterns of pyr and ths regulate mesoderm migration. (A,B,D-M) Drosophila embryo cross-sections; (A-C,D,F,H,J,L) stage 8 embryos; (E,G,I,K,M) stage 9/10 embryos. (C) Whole-mount embryo oriented with anterior to the left and dorsal up. (A) Schematic representation of a stage 8 embryo in cross-section depicting expression patterns of pyr (blue) and ths (red). (B,C) During gastrulation, pyr (blue) and ths (red) are expressed in distinct domains of the neurogenic ectoderm at the surface of the developing embryo, concurrent with spreading of mesoderm cells within the interior of the embryo. (D-G) Endogeneous pyr (D,E) and ths (F,G) expression patterns (blue). (H-M) Ectopic expression of bnl (H,I), pyr (J,K), ths (L,M) in the ectoderm using the 69B-Gal4 driver (blue).

View larger version (60K): [in this window] [in a new window]   Fig. 2. Summary of pyr and ths single-mutant isolation: genetic alleles and expression analysis. (A) The genomic region containing the Drosophila pyr and ths loci, and P-element/PiggyBac insertions and deficiencies identified in this area. Df(2R)BSC25 deletes 220 kb (Stathopoulos et al., 2004). P9.1.2 deletes 500 bp located 400 bp upstream of the ths promoter, and was achieved through a male-specific recombination screen using the P10004 insertion. Df(2R)ths238 deletes 100 kb including the entire ths coding sequence. Df(2R)pyr36 removes 100 kb, including the entire pyr coding sequence. (B-G) Double in situ hybridization using riboprobes to detect expression of both pyr (blue) and ths (red) transcripts within embryos at stage 10. Embryos are oriented with anterior to the left and dorsal up. Co-expression patterns are depicted in wild-type embryos (B-D). In pyr02915 (E) and Df(2R)pyr36 (F) mutant embryos, no pyr expression is detected, yet ths expression appears normal. In ths02026 mutant embryos, no ths expression is detected, yet the pyr expression domain appears normal (G).

View larger version (65K): [in this window] [in a new window]   Fig. 3. Pyr and Ths are both required for normal mesoderm spreading. Drosophila embryos were stained with anti-Twist antibody and sectioned as described (see Materials and methods). (A,B) Wild-type embryos; (C,D) htlAB42 mutant; (E,F) Df(2R)BSC25 mutant, which lacks both pyr and ths; (G,H) pyr single mutant (pyr02915/pyr02915); (I,J) ths single mutant [Df(2R)ths238/Df(2R)ths238]. (A,B) In wild-type embryos at stage 8, the invaginated tube collapses and the mesodermal cells start migrating (A). At stage 9/10, the spreading of the mesoderm is complete, resulting in the formation of a single layer along the internal surface of the ectoderm (B). (C-J) In mutants, the invaginated tube appears to have collapsed normally at stage 8 (C,E,G,I), but the mesoderm cells exhibit spreading defects that present as a multilayered phenotype at stage 9-10 (D,F,H,J). Cells do not appear to migrate toward dorsal regions and a monolayer is not formed.

View larger version (94K): [in this window] [in a new window]   Fig. 4. The specific expression domain of pyr is required for normal mesoderm spreading. (B,C,G,H,L,M) Drosophila embryos stained with anti-Twist antibody (brown) to detect mesoderm cells and hybridized with the specified riboprobes by in situ hybridization to detect transcripts (blue); the percentage of embryos exhibiting the mutant phenotype shown is indicated. (D,E,I,J,N,O) Embryos stained with anti-dpERK antibody. Cross-section views are of stage 9/10 embryos. (A) Schematic of a Df(2R)BSC25 mutant embryo in cross-section; no ectopic expression. (B,C) Variation in the phenotype of the Df(2R)BSC25 mutant background. Often, spreading can occur, but monolayer formation is defective (B). Alternatively, neither spreading nor monolayer formation occurs (C). (D) dpERK staining is observed in the leading edge in the wild-type embryo (see arrowhead) (Gabay et al., 1997). (E) dpERK staining is absent in Df(2R)BSC25 embryos (Stathopoulos et al., 2004). (F) Schematic of ectopic expression of either ligand (pyr or ths) in the ventral midline of Df(2R)BSC25 mutants using the sim-Gal4 driver (Xiao et al., 1996). (G-J) Ectopic expression of pyr (G,I) or ths (H,J) in the ventral midline using sim-Gal4 in the Df(2R)BSC25 mutant background. (K) Schematic of ectopic expression of either ligand within the dorsal ectoderm of Df(2R)BSC25 mutants using the zenVRE.Kr-Gal4 driver (Frasch, 1995). (L-O) Ectopic expression of pyr (L,N) or ths (M,O) in the dorsal-lateral region of the ectoderm using zenVRE.Kr-Gal4 in the Df(2R)BSC25 mutant background.

View larger version (71K): [in this window] [in a new window]   Fig. 5. The Pyr FGF ligand is necessary for the differentiation of dorsal mesoderm cell lineages. Drosophila embryos were stained with an anti-Eve antibody to examine specification of dorsal mesoderm lineages. Depicted are wild-type embryos at stage 11 (A) and stage 14 (C); Df(2R)BSC25 mutant embryos, which lack both pyr and ths genes, at stage 11 (B); pyr single mutants at stage 11 (D,E) and stage 14 (F); and ths single mutants at stage 11 (G,H) and stage 14 (I). The insets display Eve+ cell clusters at 10x magnification. (A,C) In wild-type embryos at stage 11 (A), there are 11 independent clusters of three Eve+ cells each within the dorsal somatic mesoderm. At stage 14 (C), these join to form a continuous row of heart progenitors. (B) Eve+ clusters are absent from the dorsal mesoderm of Df(2R)BSC25 mutant embryos. (D-F) In both the weakest (pyr02915/pyr02915) and the strongest [Df(2R)pyr36/Df(2R)pyr36] alleles of pyr single mutants (D and E, respectively), the number of Eve+ clusters is significantly reduced, resulting in gaps within the row of heart progenitors at stage 14 (F). (G-I) By contrast, within ths mutant embryos at stage 11, when either a weak mutant [Df(2R)ths238/ths02026] or the strongest alleles [Df(2R)ths238/Df(2R)ths238] are examined (G and H, respectively), there are only subtle effects on Eve+ cell specification (see inset; often two Eve+ cells are present instead of three). At later stages, defects are more apparent (I).

View larger version (62K): [in this window] [in a new window]   Fig. 6. Pyr and Ths, but not Bnl, can support Htl activation to effect expression of Eve within dorsal mesoderm lineages. Drosophila embryos oriented with anterior to the left and dorsal up. Ectopic expression of UAS-pyr, UAS-ths or UAS-bnl in a Df(2R)BSC25 mutant background, which lacks the endogenous pyr and ths genes, was achieved using various Gal4 drivers that support expression in different domains of the ectoderm. Depicted are lateral views of stage 11 embryos stained using an anti-Eve antibody (green) and by in situ hybridization with riboprobes to detect pyr (D,G), ths (A-C,E,H) or bnl (F,I) transcript levels (red). (A) Endogeneous expression of ths in wild-type embryos. Eve+ cells are present in 12 hemisegments. (B) ths expression in Df(2R)BSC25 mutant embryos. No dorsal mesoderm-derived Eve+ cells are present. Eve staining is detected only in the central nervous system, expression that is FGF-signaling independent. (C) Expression of ths in the ventral midline using sim-Gal4 does not support expression of Eve in a homozygous Df(2R)BSC25 mutant background. (D-F) Ectopic expression of pyr (D) or ths (E) in the ectoderm using 69B-Gal4 does support expression of Eve+ in a homozygous Df(2R)BSC25 mutant background, whereas bnl (F) does not. (G-I) Ectopic expression of pyr (G) or ths (H) in the ectoderm using zenVRE.Kr-Gal4 also supports expression of Eve+ in a homozygous Df(2R)BSC25 mutant background, whereas again bnl (I) does not.

View larger version (30K): [in this window] [in a new window]   Fig. 7. A model for FGF signaling through Heartless. (A) The location of the Ths and Pyr expression domains is important for the proper regulation of mesoderm migration. Both ligands are required, presumably because they have different activities. (B) During specification of dorsal mesoderm lineages, including specification of Eve+ cells (orange), FGFs feed into an array of signaling molecules [Wingless (Wg) and Dpp] necessary to specify dorsal mesoderm lineages. We suggest that any FGF ligand (red), expressed in the region of Dpp and Wg overlap and able to activate the respective FGFR, would suffice to support cell differentiation. Wg, Wingless.

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