p38 mitogen-activated protein kinase can be involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis

Abstract

p38 mitogen-activated protein kinase (p38) has been extensively studied as a stress-responsive kinase, but its role in development remains unknown. The fruit fly, Drosophila melanogaster, has two p38 genes, D-p38a and D-p38b. To elucidate the developmental function of the Drosophila p38's, we used various genetic and pharmacological manipulations to interfere with their functions: expression of a dominant-negative form of D-p38b, expression of antisense D-p38b RNA, reduction of the D-p38 gene dosage, and treatment with the p38 inhibitor SB203580. Expression of a dominant-negative D-p38b in the wing imaginal disc caused a decapentaplegic (dpp)-like phenotype and enhanced the phenotype of a dpp mutant. Dpp is a secretory ligand belonging to the transforming growth factor beta superfamily which triggers various morphogenetic processes through interaction with the receptor Thick veins (Tkv). Inhibition of D-p38b function also caused the suppression of the wing phenotype induced by constitutively active Tkv (TkvCA). Mosaic analysis revealed that D-p38b regulates the Tkv-dependent transcription of the optomotor-blind (omb) gene in non-Dpp-producing cells, indicating that the site of D-p38b action is downstream of Tkv. Furthermore, forced expression of TkvCA induced an increase in the phosphorylated active form(s) of D-p38(s). These results demonstrate that p38, in addition to its role as a transducer of emergency stress signaling, may function to modulate Dpp signaling.

FIG. 1

Complementation of the S. cerevisiae MAPK hog1Δ mutant by Drosophila members of the MAPK superfamily. The D-p38b⁺ clone clearly complements the high-osmolarity (0.9 M sorbitol)-sensitive growth phenotype of the hog1Δ mutant, while the DJNK⁺ clone does so only weakly. The Rl⁺ clone fails to complement this phenotype. When the TGY sequence of D-p38b, a putative MAPKK phosphorylation site, was mutated (T183A and Y185F), complementation was abolished.

FIG. 2

Exon organization and mapping of the D-p38b gene. (A) Exon organization of the D-p38b gene. Two exons are indicated by boxes. The coding region is filled. Nucleotide 1 is assigned to the 5′ end of the longest D-p38b cDNA. (B) In situ chromosomal hybridization. Salivary gland polytene chromosomes were derived from the hemizygote of Df(2L)b82a2 which had lost the region from 34D1 to -2 through 34E1 to -2 (37). The hybridization signal (arrow) was observed at the 34D region. This region expands somewhat owing to the compression caused by pairing of the wild-type chromosome with the deficiency chromosome. The method used for digoxigenin-labelled in situ hybridizations has been described previously (2).

FIG. 3

Effect of D-p38b^DN on wing phenotype. (A) Wild-type Canton-S. L2, L3, L4, and L5 indicate the four longitudinal veins. (B) An individual expressing high levels of D-p38b^DN. The distance between L4 and L5 (double-headed arrow) is reduced, similar to what is seen in the dpp mutant (panel C). Ectopic veins around distal L2 (arrow) are reminiscent of those in various tkv mutants (see panel D) (13, 37, 45). A notched wing margin is occasionally observed with other dpp alleles (see panel F). (C) An example of a very hypomorphic dpp allele (dpp^d5/dpp^hr27) (37). (D) An example of a hypomorphic tkv allele (tkv⁷/tkv⁴²⁷). (E) A dpp mutant expressing moderate levels of D-p38b^DN. L4 and L5 were partially fused, and the distance between L2 and L3 was also reduced, similar to that seen with the severe dpp alleles (9) (see panel F). (F) An example of a severe dpp allele (dpp^d5/dpp^hr56) (37).

FIG. 4

Effects of reduced Mad gene dosage, the p38 inhibitor SB03580, and reduced D-p38 gene dosage on the adult wing phenotype induced by constitutively active Tkv (Tkv^CA). (A through F) Adult wing phenotypes. All these wings are from individuals carrying one copy each of UAS-tkv^CA-S and 71B-GAL4. (A) Wild-type background for other genes. Incision of wing margins was frequently observed. (B) Hemizygote for Df(2L)C28 (49) that uncovers the Mad locus. Reduction of the Mad gene dosage suppresses the tkv^CA wing phenotype. (C) An individual fed a standard diet supplemented by 120 nM SB203580 (a p38 inhibitor; Calbiochem/Novabiochem). SB203580 suppresses the tkv^CA wing phenotype. (D) Hemizygote of Df(3R)crb-F89-4 that uncovers the D-p38a locus. Reduction of D-p38a gene dosage does not suppress the tkv^CA wing phenotype. (E) Hemizygote of Df(2L)b82a2 that uncovers the D-p38b locus. Reduction of D-p38b gene dosage suppresses the tkv^CA wing phenotype. (F) Hemizygote of Df(2L)b82a2 carrying one copy of UAS-D-p38b⁺. Suppression by reduced D-p38b gene dosage was abrogated by reintroducing the D-p38b⁺ transgene. (G) Quantitative representation. Histograms represent percentages of wings in which L5 is detached from the wing margin.

FIG. 5

Effects of altering D-p38b function and reducing DJNK, Dpp, and Tkv functions on the wing phenotypes caused by either UAS-tkv^CA-W (W) or UAS-tkv^CA-S (S) driven by 71B-GAL4. (A through F) Adult wing phenotypes. All these wings are from individuals carrying one copy each of UAS-tkv^CA-S and 71B-GAL4. (A) Wild-type background for other genes. (B) An individual carrying one copy of UAS–D-p38b^antisense. D-p38b^antisense suppresses the tkv^CA wing phenotype. (C and D) Individuals carrying one copy (C) or two copies (D) of UAS–D-p38b^DN-W. D-p38b^DN suppresses the tkv^CA wing phenotype in a dose-response manner. (E) An individual carrying two copies of UAS–D-p38b^DN-W together with one copy of UAS–D-p38b⁺. Suppression by D-p38b^DN was abrogated by coexpression of D-p38b⁺. (F) Heterozygote of dpp^d8 (37). Reduction of the dpp gene dosage does not suppress the tkv^CA wing phenotype. (G) Quantitative representation. Histograms represent percentages of wings in which L5 is detached from the wing margin. ++, two introduced copies of the transgenes. Df(DJNK), Df(2L)flp147E (46, 50).

FIG. 6

D-p38b regulates early Dpp-Tkv signaling-dependent omb expression in the wing disc. (A) The omb expression pattern visualized by a lacZ reporter, omb^P1 (51). Anterior is to the top and dorsal to the left. (a) omb^P1/+; 69B-GAL4/+ (control). (b) omb^P1/+; 69B-GAL4/UAS-tkv^CA-S. Tkv^CA induces ectopic expression of omb (arrow) and overgrowth. This photograph is reduced to 75% the size of the others. (c) omb^P1/UAS-D-p38b^DN-W; 69B-GAL4/UAS-tkv^CA-S. (d) omb^P1/UAS-D-p38b^antisense; 69B-GAL4/UAS-tkv^CA-S. Expression of either D-p38b^DN or D-p38b^antisense reduced Tkv^CA-induced ectopic omb expression (arrows) and overgrowth (c or d, respectively). (B) D-p38b^DN-expressing clones generated outside the domain in which dpp is expressed display reduced sensitivity of omb induction to Tkv^CA. Note that dpp expression at this stage in the wing disc lies in a narrow belt just anterior to the anteroposterior boundary (20, 24). Clones of cells that expressed various UAS-transgenes under the control of Actin5C-GAL4 were generated by the flp-out technique and marked by the presence of green fluorescent protein (GFP) (28) (a and d, green). omb expression was revealed by staining with an antibody raised against β-galactosidase (Promega) (b and e, red). (a through c) Wing disc-carrying clones expressing both D-p38b⁺ and tkv^CA as controls (hsFlp/omb^P1 UAS-D-p38b⁺; AyGAL4 UAS-GFP/+; UAS-tkv^CA-S/+). (d through f) Wing disc-carrying clones expressing both D-p38b^DN and tkv^CA (hsFlp/omb^P1 UAS-D-p38b^DN-S; AyGAL4 UAS-GFP/+; UAS-tkv^CA-S/+). (c and f) Superimposed images.

FIG. 7

Enhancement of omb^bi by D-p38b^DN or by D-p38b^antisense. (A) A wing of an omb^bi/Y male. Arrows indicate the aberrations typically observed: fusion of veins at the base of the wing and small notching at the wing tip. (B) A wing of an omb^bi individual expressing D-p38b^antisense (omb^bi/Y; UAS-D-p38b^antisense/69B-GAL4). (C and D) Wings of omb^bi individuals expressing D-p38b^DN weakly (omb^bi UAS–D-p38b^DN-W/Y; 69B-GAL4/+) (C) or strongly (omb^bi UAS–D-p38b^DN-S/Y; 69B-GAL4/+) (D). Arrows point to the omb^bi phenotypes.

FIG. 8

Phosphorylation and activation of D-p38b. (A) D-p38b is tyrosine phosphorylated after heat shock. Extracts from adult flies left untreated or treated with heat (37°C for 1 h) were immunoprecipitated (IP) with anti-p-Tyr antibody (α-p-Tyr) and immunoblotted with anti-D-p38b antibody after separation by SDS-polyacrylamide agarose gel electrophoresis (PAGE) (upper panel). Total extracts were also immunoblotted with anti-D-p38b antibody (lower panel). In this experiment, flies carrying one copy each of UAS-D-p38b⁺ and hs-GAL4 (6) were used to facilitate detection. The total amount of D-p38b did not change immediately after heat shock. The basal activity of the heat shock promoter was sufficient to achieve constitutive expression of GAL4; thus, D-p38 was overexpressed even in the absence of heat treatment. (B) Anti-p-p38 antibody recognizes phosphorylated D-p38b in fly extract. Flies harboring both UAS-D-p38b⁺ and hs-GAL4 (right half) or only hs-GAL4 (left half) were heat shocked as described above, and extracts were immunoblotted with either anti-p-p38 or anti-D-p38b antibody after separation by SDS-PAGE. The intensities of bands recognized by anti-p-p38 increased as a result of either heat treatment or D-p38b overexpression. (C) The phosphorylation level of D-p38b correlates with its enzymatic activity. Recombinant D-p38b protein was pretreated with recombinant MKK6 in the presence of cold ATP. Then ATF2 and [γ-³²P]ATP were added to aliquots of reaction mixtures, and incubation was continued. The resulting mixtures were resolved by SDS-PAGE and subjected to autoradiography for detection of kinase activities (top panel) or to immunoblotting with anti-p-Tyr and anti-p-p38 antibodies (two middle panels). The bottom panel represents the gel stained with Coomassie brilliant blue (CBB). The kinase assay method used has been described elsewhere (30). (D) Activation of D-p38(s) by Tkv^CA. Extracts from larvae of Canton-S (wild type) or from larvae carrying two copies each of UAS-tkv^CA-S and 71B-GAL4 were resolved by SDS-PAGE and immunoblotted with anti-p-p38 or anti-D-p38b antibody.