BIOLOGICAL OVERVIEW

Mitogen activated protein kinases or MAP kinases (MAPKs) are members of relay systems involving multiple, interacting cascades of serine/threonine phosphorylations. These proteins are responsible for passing information from the cell surface (in response to mitogens) to the nuclei, stimulating new gene expression. Based on genetic, structural and biochemical studies, three main MAPK pathways can be distinguished: the ERK, JNK, and p38 pathways. Genetic studies in invertebrates have demonstrated clearly an essential function for the ERK (extracellular signal-related kinase) and JNK [Jun amino (N)-terminal kinase)] pathways during development. However, although it has been shown that p38 signaling has a role in apoptosis and is activated by many environmental stress signals, a function for p38 signaling in normal development has not yet been established. The topic of this overview, the gene licorne (lic), is a MAPK kinase, targeting the MAPK known as p38.

To address the role of p38 signaling in Drosophila development, licorne (French for unicorn), a Drosophila p38 MAPKK (read MAP kinase kinase) was isolated. Both in yeast and in cell cultures, lic can specifically activate vertebrate p38, suggesting that at least some components of the p38 pathway are conserved in Drosophila. The isolation and phenotypic analyses of lic mutations indicate an essential and complex lic function during oogenesis (Suzanne, 1999).

In Drosophila two inductive events originating from the oocyte and directed toward the overlying follicle cells account for the sequential establishment of the anterior/posterior (AP) and dorsal/ventral (DV) axes during oogenesis. The oocyte signal is encoded by the TGF-alpha gurken gene, whose products are tightly associated with the nucleus and move from an initial posterior to an anterodorsal position during oogenesis. grk first activates the Epidermal growth factor receptor (Egfr) at the posterior most region of the oocyte and subsequently at the anterior dorsal region of the oocyte, signaling to overlying follicle cells, determining their fate. One consequence of posterior follicle cell determination is the sending back of an as-yet unknown signal to the oocyte, leading to microtubule repolarization, a nucleus anteriorward migration and asymmetric localization of the maternal determinants bicoid, oskar, and gurken. When the oocyte nucleus reaches the anterodorsal corner of the oocyte, a second peak of grk activity determines dorsal follicle cells fate and thus influences the DV axis of both the eggshell and the embryo (for reviews, see Ray, 1996 and Anderson, 1998).

licorne mutations provoke polarity defects in the eggshell and embryo, as a result of reduced activity of two localized determinants: osk and grk. The phenotypic similarities between lic and grk mutations suggest that lic patterning defects arise from a reduction of grk activity at the posterior and dorsal poles of the oocyte, placing lic in a pathway required for post-transcriptional regulation of grk. Thus the p38 MAPK pathway is essential for oogenesis. Unravelling the role of p38 MAPK in oocyte asymmetry will help in understanding the complex interplay between multiple factors required for Drosophila axis determination (Suzanne, 1999).

licorne was first cloned on the basis of homology with mammalian p38 homologs (S. Han, 1998 and Z. Han, 1998). A subsequent publication reported the cloning of lic on the basis of the gene's ability to complement a defect in MAPKK in yeast. The yeast hog1 pathway mediates cellular responses to an increase in external osmolarity (Herskowitz, 1995). Mammalian p38 has been shown to complement the high osmolarity-sensitive growth phenotype of the hog1 MAPK mutant. This complementation is dependent on the presence of pbs2, a MAPKK that functions as an upstream activator of hog1. These data suggest that the pbs2 MAPKK is required for the activation of mammalian p38 in yeast, raising the possibility that yeast could be used to identify the Drosophila MAPKK(s) activating p38. A polymyxin B sensitivity (pbs) pbs2Delta mutant expressing p38 was transformed with a Drosophila cDNA library and screened for growth on medium containing 1 M sorbitol (high osmolarity). Out of a total of 20 transformants capable of growth in the presence of sorbitol, six clones failed to restore high-osmolarity resistance in the absence of p38 expression, indicating that these transformants suppressed the pbs2Delta mutation in a p38-dependent manner. These six candidates define two classes, as determined by restriction enzyme analysis, and one class represents clones of licorne (Suzanne, 1999).

licorne mutant embryos are defined, for the purpose of this study, as hemipterous;licorne double mutants engineered to express a hemipterous transgene (see Effects of Mutation for more information about this genotype). lic mutant embryos show a segmentation phenotype that is reminiscent of the one produced by mutations in the maternal posterior-group genes, including oskar, vasa, and nanos. Most of the posterior-group genes can provoke both abdominal segmentation defects and a loss of germ cells, a dual defect that is due to the common localization of the posterior and germ cell determinants in the posterior germ plasm. Like several posterior-group genes, lic embryos lack or have a strongly reduced number of pole cells, as shown using Vasa and Nanos as markers. In most lic mutant embryos, Vasa protein fails to be accumulated at the posterior pole, although in some cases weak staining is observed. It is concluded that lic has a role in abdominal segmentation, proper Vasa protein and Nanos mRNA localization at the posterior pole, and formation of the pole cells. These results suggest that lic also has a role in germ plasm assembly (Suzanne, 1999).

The assembly of the germ plasm takes place during oogenesis and proceeds in several steps leading to the successive posterior localization of many different components (for review, see Rongo,1996). A pivotal step in this process is the localization of the OSK mRNA to the posterior pole of the oocyte in stage 8-9 egg chambers, which is the basis for the recruitment and assembly of downstream components like Vasa and Nanos. In lic germ-line clones, both OSK mRNA expression and early posterior localization appear normal until stage 8 of oogenesis. However, in stage 9 and older egg chambers, the OSK mRNA is mislocalized, diffusing in the whole oocyte in a gradient from the posterior to the anterior pole. In later stages, OSK transcripts are barely detectable, indicating that diffusion proceeds continuously in mutant egg chambers. A similar phenotype is observed in some osk missense mutants, suggesting a role for Osk protein in the anchoring of its own mRNA at the posterior pole. Staining of lic mutant egg chambers using an anti-Osk antibody did not allowed detection of any reduction in Osk protein accumulation, indicating that lic affects OSK mRNA localization independent of Osk translation (Suzanne, 1999).

In some lic egg chambers, the mislocalized OSK mRNAs also seem to partly accumulate in a more central position, reminiscent of the position of OSK transcripts in mutants that have not reorganized the microtubules, as in EGFR pathway mutants. This result suggests that lic oocytes are not completely repolarized. However, no defect in the positioning of the nucleus, or in the localization of a kinesin-lacZ microtubule-associated motor protein fusion is observed, suggesting that OSK mRNA mislocalization is a more sensitive assay and lic defects are weak. The correct localization of osk RNA at stage 8 and its later diffusion indicate that lic affects the maintenance of OSK mRNA asymmetric localization in the oocyte (anchoring) rather than the mechanism of localization per se, most likely as a result of incomplete polarization along the AP axis (Suzanne, 1999).

In addition to its requirement in AP patterning, lic mutations also affect the DV axis, as evidenced by ventralization of the eggshell. One important event in DV patterning is the correct localization of the Gurken ligand on the future dorsal side of the oocyte, a position that depends on the correct localization of the nucleus in the oocyte. Because mislocalized nuclei was never observed in lic mutant oocytes, lic DV defects are not likely to be the result of inappropriate nucleus migration or microtubule polarization. It was thus asked whether the grk determinant itself might be affected in lic mutants. In situ hybridization using a grk probe did not detect any defect, suggesting that expression and localization of the GRK mRNA are normal in lic mutant oocytes. However, immunostaining of egg chambers using an anti-Grk antibody shows reduction (15%) or mislocalization (~5%) of Grk protein. To further characterize a loss of grk activity in lic oocytes, wild-type and mutant ovaries were stained using a kekkon (kek)-lacZ reporter construct. The kek gene is a target of the Egfr in the follicle cells and thus serves as an indirect and sensitive assay to measure grk activity in the oocyte. In wild-type egg chambers, kek is expressed in dorsal follicle cells in a characteristic graded pattern reflecting both the intensity and localization of the underlying grk signal. In ~50% stage 10 lic mutant egg chambers, kek expression is reduced dramatically, as shown by a reduction in the number of responding follicle cells and a change in the shape of the kek expression domain. In rare cases (<5%), an expansion of the kek signal in more lateral and ventral positions is also observed, an observation that might suggest a partial delocalization of grk activity in the oocyte. Consistent with this result, dorsalization of the chorion is observed in very rare cases. Thus, lic loss of function in the germ line reduces Egfr activity in the dorsal follicle cells, most likely as a result of a reduction of grk activity in the oocyte (Suzanne, 1999).

The requirement for lic function in the oocyte raises the question of how p38 is activated. lic phenotypes are observed in the oocyte, an unusual cell in the sense that its nucleus is mostly transcriptionally silent. So far, all MAPK pathways have been shown to modulate gene activity in response to several stimuli. If lic does regulate transcription in the germ line, then a likely possibility is that these nuclear events take place in the nurse cells and that newly expressed gene products are transmitted to the oocyte through the ring canals. This may represent a unique and novel mechanism of MAPK signaling (Suzanne, 1999).

A series of elegant genetic studies have demonstrated a clear link between the establishment of the AP and DV axes and the activation of the Egfr in specific follicle cells. These patterning activities rely on cell communication between two different cell populations in the egg chamber: the germ-line cyst and the surrounding somatic follicle cells. Egfr activation in the follicle cells is triggered by the activity of Grk in the underlying oocyte, and farther downstream signal transduction is mediated by the well-characterized ras/raf/ERK MAPK pathway. The p38 MAPK pathway also participates in asymmetric development of the egg chamber. Interestingly, ERK and p38 signaling are not active in the same cells, because lic function is restricted to the germ line, whereas ERK is only active in the follicle cells. The loss of function of lic or ERK lead to similar DV phenotypes, suggesting an interaction between these two MAPK pathways. This view is well supported by the observation that grk activity and localization are affected in lic mutants. Altogether, these results indicate that patterning of the egg relies on the activation of both the p38 and ERK pathways, representing the first example of cell communication based on activation of two different MAPK pathways in two distinct and apposing cell types (Suzanne, 1999 and references).

The lic/Dp38K and hep/DJNKK genes are clustered in the same locus, suggesting that these related genes may have a common ancestral origin. Studies in mammalian cells have shown that p38 and JNK pathways can be activated by identical stress stimuli (for review, see Kyriakis, 1996), indicating that these related pathways might also work together in some developmental processes. However, the functional analysis of hep and {hep;lic} mutants show that each JNKK and p38 MAPKK gene has derived specific functions, leading to specific loss-of-function phenotypes. hep controls morphogenesis of the lateral ectoderm during dorsal closure of the embryo, without affecting AP patterning (Glise, 1995 and Glise, 1997). This study shows that lic loss of function mutations affect asymmetric development during oogenesis, with phenotypic consequences on DV and AP patterning of the eggshell and the embryo. Because lic and hep are involved in different processes and the double mutant leads to additive phenotypes, it thus appears that in Drosophila the JNK and p38 MAPK pathways act independently during embryonic development. However, it is still possible that, as in vertebrates, stressful stimuli may be able to activate both Drosophila p38 and JNK pathways, an issue that can now be addressed genetically using lic and hep mutations (Suzanne, 1999).

GENE STRUCTURE

lic is located on the X chromosome, in close proximity to the hemipterous (hep) gene (11D1-2 on the cytological map). hep encodes a MAPKK of the JNKK family involved in epithelial morphogenesis during dorsal closure of the embryo (Glise, 1995).

Bases in 5' UTR - 360

Exons - 5

Bases in 3' UTR - 602

PROTEIN STRUCTURE

Amino Acids - 335

Structural Domains

Degenerate oligonucleotides were used to amplify MKK-related sequences from a Drosophila embryonic cDNA library, using PCR. Two novel protein kinases were identified. Sequence analysis of full-length cDNAs led to the identification of homologs of human MKK3 and MKK4 (H-MKK3 and H-MKK4). The sequence of D-MKK3 (Licorne) is 53% identical to that of H-MKK3. The identity between D-MKK4 and H-MKK4 is 50%. Analysis of the genomic sequences has demonstrated that both D-MKK genes contain a small number of introns in the coding region (S. Han, 1998).

Lic protein is related to the MAPKK family, with strongest sequence identity to human p38 activators MKK3 and MKK6 (Derijard, 1995 and Lin, 1995). The two known phosphorylation sites required for MAPKK activation are well conserved in Lic, leading to the conclusion that lic encodes a Drosophila MAPKK of the p38 MAPKK family. Recently, the Drosophila Genome Project and PCR-based approaches have allowed the identification of DMKK3 (Licorne), a Drosophila homolog of human MKK3 and MKK6, and two related p38 protein kinases. On the basis of sequence identity and chromosomal location, it is concluded that DMKK3 and lic correspond to the same gene (Suzanne, 1999).

date revised: 20 July 99

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