The Drosophila p38 MAPK pathway is required during oogenesis for egg asymmetric development

Abstract

In mammalian cells, the p38 mitogen-activated protein kinase (MAPK) pathway is activated in response to a variety of environmental stresses and inflammatory stimuli. However, the role of p38 MAPK signaling in unchallenged conditions remains largely unknown. We have isolated mutations in a Drosophila p38 MAPKK gene homolog, licorne (lic), and show that during oogenesis, lic is required in the germ line for correct asymmetric development of the egg. In lic mutant egg chambers, oskar mRNA posterior localization is not properly maintained, resulting in anteroposterior patterning defects in the embryo. Furthermore, lic loss-of-function in the germ line leads to reduced EGF receptor activity in dorsal follicle cells and ventralization of the egg shell. Both these defects are associated with a diminution of gurken protein levels in the oocyte. Our phenotypic data argue for a role of lic in a post-transcriptional regulation of the grk gene. Furthermore, they show that in addition to the well-characterized Ras/Raf/ERK MAPK pathway acting in the follicle cells, another related signaling cascade, the p38 MAPK pathway, is required in the germ line for correct axes determination. These results provide the first genetic demonstration of an essential function for a p38 pathway during development.

Figure 1

Characterization of the lic gene. Complementation of a pbs2 deletion mutant in Saccharomyces cerevisiae using lic cDNA. (A) Yeast strain TM334 (pbs2) was transformed with various plasmids as indicated. Transformants were streaked onto YPD plates containing 1 m sorbitol and incubated at 30°C. (B) Yeast strain TM334 (pbs2) was transformed with empty vector (−) or an expression vector for HA–LIC (+). Cells were treated with NaCl (0.5 m) for the indicated times. Immunoprecipitated complexes obtained with anti-HA were used for in vitro kinase reactions with GST–p38-KN as a substrate (top). The amounts of immunoprecipitated HA–LIC were determined with anti-HA (bottom). (C) Complete sequence of full-length lic cDNA (accession no. AJ238572) and a phylogenetic tree showing the relationship between lic and other Drosophila and human MAPKKs. lic is most homologous to the human MKK3 and MKK6 p38 activators (D).

Figure 2

Molecular organization and expression of lic. (A) Genomic organization of the {hep, lic} locus. hep1 is a mobile P element inserted in the hep 5′ untranslated region, and was used to generate double {hep, lic} mutant alleles by imprecise excisions. The lines above the restriction map indicate the DNA lesions associated with five {hep, lic} alleles, as determined by Southern blot analysis. The solid boxes indicate DNA deletions within the two adjacent EcoRI fragments covering hep and lic. Below the restriction map is represented a schematic structure of full-length lic cDNA. Broken lines indicate the introns; solid boxes represent the coding region. (E) EcoRI. lic is expressed throughout development at variable levels. Expression in early (0–2 hr) embryos indicates a maternal contribution. The same blot was hybridized using a ribosomal gene probe (rpL17A) to assess mRNA levels. (C) In situ hybridization to wild-type ovaries indicates germ-line expression in nurse cells from stage 7–8 onward. (D) In {hep, lic}H6 germ-line clones, lic mRNAs are not detected. lic expression in the germ line can be restored using a UBlic transgene as shown in E.

Figure 3

Chorion and cuticular phenotypes of lic. Dark-field photographs of chorion (A,B) and embryo cuticles (C–E) preparations of wild type (A,C) and lic eggs or embryos derived from homozygous {hep, lic} germ-line clones (B,D,E). The chorion in B shows a ventralized phenotype as stated by fusion of dorsal appendages. (D) Partial deletion of the A4 abdominal segment indicates a weak abdominal segmentation defect. (E) In extreme cases, the whole abdominal region is deleted and eventually replaced by a single large denticle band. The terminal structures like the head skeleton and the Filzkörpers are normal. Anterior is on the left.

Figure 4

lic mutations affect pole plasm assembly. Immunostaining of wild-type (A,B), {hep, lic} (D–F), and {hep, lic; UBlic} (C) embryos using an anti-vasa antibody. In wild type, the vasa protein is localized in a posterior crescent in early embryos (A) and becomes incorporated into the pole cells once they form (B). In lic mutant embryos, no (D) or little (F) vasa is present at the posterior pole, and no pole cells form (E). These defects are due to a loss of lic function, as expression of a lic cDNA in the germ line using a UBlic transgene can restore vasa expression and posterior localization (C). Anterior is on the left.

Figure 5

lic is required for the maintenance of osk mRNA posterior localization in the oocyte. In situ hybridization of wild-type (A,B) and lic mutant (C–E) oocytes at stages 9 (A,C,D) and 12 (B,D) of oogenesis. (A,B) In wild type, osk mRNA is localized posteriorly in the oocyte until the end of oogenesis. (C) In lic mutant egg chambers, osk mRNA normally localizes at the posterior pole of the oocyte but is not properly maintained and diffuse toward more anterior regions. (Arrowhead in D) In some cases, osk mRNA also accumulates centrally. (Arrowhead in E) osk transcripts are barely detectable in stage 12 oocytes, indicating that diffusion of osk proceeds continuously throughout oogenesis. Anterior is on the left.

Figure 6

lic controls grk activity in mid-oogenesis. Immunostaining of wild-type (A) and lic (D,G) stage 9 oocytes using an anti-grk antibody (lateral views; dorsal is up). (D) In mutant oocytes, grk protein is mislocalized. The egg chamber in G represents the most extreme localization defect that we have observed. β-Galactosidase staining of wild type (B) and lic (E,H) stage 10 egg chambers shows kek expression in the dorsal region of the follicle cells (45%; n = 49). Note that in lic mutants, kek expression is affected, both in terms of levels and shape of the expression domain. In rare cases, kek expression is expanded laterally and ventrally, suggesting a dorsalization of the future egg chorion (I; <5%). (C,F,I) The chorion phenotypes corresponding to grk and kek expression patterns in wild-type (C) and lic egg chambers (F,I; 50% and <1%, respectively; n = 219). B, C, E, F, and I are dorsal views. H is a lateral view, with dorsal up. For all panels, anterior is on the left.

Figure 7

Model of p38 MAPK pathway function during oogenesis. The germ line (nurse cells and the oocyte), the site of lic/p38 activity, is shaded; dots show the sites of EGFR activity in the follicle cells (posterior and dorsal). (See Discussion for details.)