licorne

DEVELOPMENTAL BIOLOGY

Embryonic

The expression pattern of D-p38b is very similar to that of the two D-MKKs (Licorne and DMKK4), which also have high maternal deposition in early embryos and zygotic expression in the midguts. One noteworthy difference between the two D-MKKs is that D-MKK4 expression is sustained in the ventral nerve cord, while Licorne expression is less detectable in this tissue but is more prominent in the midgut. Previous studies have demonstrated that D-JNK expression is present in the ventral nerve cord, suggesting a functional relationship with D-MKK4 in this tissue. Since each of these kinases is deposited maternally, they may all be involved in early development. At later stages, D-p38b may be the primary mediator of the p38 MAPK pathway, particularly in the developing midgut (Z. Han, 1998).

Northern blot analysis and whole-mount in situ hybridization to embryos using a lic probe indicate a strong maternal contribution. In ovaries, LIC mRNAs are detected in the germ line from stage 7-8 onward. Expression is found in the nurse cells and mRNA accumulates in the oocyte by stage 10-11, although clear mRNA accumulation in the oocyte is only visible in later stages (Suzanne, 1999 and references).

Effects of Mutation or Deletion

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

Mutations in a Drosophila p38 MAPKK gene homolog, licorne (lic), have been isolated. 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. These 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 (Suzanne, 1999).

Double mutants of hemipterous and licorne (hep;lic) were generated by imprecise excision of a P element inserted into the hep gene, which is closely linked to the lic gene. The maternal expression of lic prompted a study its function using germ-line clonal analysis of hep;lic double mutations. The hep and or lic mutations can be compensated for by expression of hep or lic respectively from a Ubiquitin promoter (UBhep or UBlic respectively). To determine the phenotype(s) contributed by lic mutations, a comparative analysis was made of hep;lic germ-line clones induced in females expressing either UBhep;UBlic, or both UBhep and UBlic transgenes. Cuticle preparations of embryos laid by hep;lic or hep;lic;UBhep germ-line clones (hereafter referred to as lic embryos) show novel and complex phenotypes, as compared to those generated using a simple hep mutation. Most of the eggs (80%-90%) appear not to be fertilized, are round in shape, and show a smaller size than the wild type, suggestive of incorrectly polarized eggs. In addition, the dorsal appendages, which mark the dorsal anterior region of the eggshell, are often shifted to a more dorsal position (50%); in ~20% of the eggs, dorsal appendages are fused, indicating a weak ventralization of the eggshell, reminiscent of some Egfr pathway hypomorphic mutants like grk and the Egfr. Finally, analysis of mutant egg chambers showed a highly penetrant 'dumpless' phenotype, that is, improper transfer of nurse cell content into the oocyte at the end of oogenesis, a defect that may account for the reduced size of laid eggs (Suzanne, 1999).

Cuticle preparations of developed embryos (10%-20%) show variable but characteristic segmentation defects, consisting in the progressive deletion of abdominal segments (ranging from a partial deletion of segment A4 to a complete deletion of abdominal segments). This phenotype is reminiscent of that of the posterior-group genes, which control abdominal and pole cell development. Because of the presence of a double mutation, some embryos show both a dorsal hole (due to the dorsal closure function of hep) and a deleted abdomen, indicative of an additive phenotype of the two MAPKK mutations. When clones are induced in females expressing a UBhep transgene, the dorsal opening is completely rescued, confirming the hep origin of this phenotype. However, the unfertilized, ventralized, and segmental defects remain. In contrast, in embryos derived from hep;lic;UBlic clones, the proportion of unfertilized eggs decreases strongly (from 80% to 30%), and eggshell ventralization defects or embryonic segmentation are no longer observed, even though the proportion of developing embryos is greatly increased. These results indicate that the complex phenotypes associated with hep;lic chromosomes, including unfertilization and patterning defects, are a specific effect of lic loss of function (Suzanne, 1999).

One puzzling observation is that the polarity defects associated with lic mutations are not fully penetrant (80% of embryos show vasa localization defects; 50% of egg chorions show a ventralization phenotype), despite the fact that the lic mutants described in this study are likely to be null alleles. Although variable phenotypes are commonly observed among oogenesis genes, their origin is not well understood. In the case of lic, which belongs to the stress-activated family of MAPK pathways, it is possible that its loss of function will become more dramatic in stressful conditions, a view that is in part supported by the fact that lic phenotypes are cold sensitive. Another, nonexclusive possibility is that partial redundancy may occur. This is supported by the fact that two different Drosophila p38-encoding genes have been identified, Dp38a and Dp38b (S. Han, 1998), a situation that is unique in flies, since only one MAPK was identified that functions in the other ERK and JNK pathways. Given that only one p38 activator gene has been cloned so far (lic/DMKK3), the existence of another related function, and/or the possibility that lic may only activate one Dp38 MAPK in vivo, cannot be exclused. A way to address these issues will be to compare Dp38a and Dp38b single and double mutant phenotypes, once these become available (Suzanne, 1999).

The Drosophila STE20-like kinase Misshapen is required downstream of the Frizzled receptor in planar polarity signaling.

Misshapen acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, evidence is provided for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn (Paricio, 1999).

Although there is accumulating evidence that JNK-type MAPK modules are involved in planar polarity signaling, the analysis of mutant clones of either hep or bsk alleles shows no or weak phenotypes in imaginal discs. These observations suggest a high degree of redundancy at this level in the polarity signaling pathway. To address this issue further, a potential involvement of related kinases that could account for the proposed redundancy was examined. The recently described Drosophila kinases, belonging to the JNK/p38 class within the MAPK modules were examined for genetic interactions with the planar polarity phenotypes of sev-Dsh and sev-msn. These are obvious candidates to be cooperating with Hep and Bsk in polarity generation. At the level of Hep/JNKK (an MKK7 homolog), two other MKKs have been reported (DMKK3 and DMKK4). Similarly, at the level of Bsk/JNK, two p38-like kinases were isolated (Dp38a and Dp38b). Since no mutants have yet been isolated for these genes, whether deficiencies removing these kinases would show an interaction with sev-Dsh was examined. DMKK3 maps in the vicinity of hep: deficiencies removing DMKK3, Df(X)G24 and Df(X)H6, also remove hep. These deficiencies show externally a very strong suppression of sev-Dsh with a marked decrease of misrotated ommatidia as observed in tangential sections. Deficiency Df(3R)p13 removes the DMKK4 locus and also dominantly suppresses sev-Dsh. Similarly, deficiencies removing either Dp38a, Df(3L)crb^87-4 and Df(3L)crb^F89-4, or Dp38b, Df(2L)b80e3 and Df(2L)b87e25, are suppressors of sev-Dsh. Whether the respective deficiencies showed an interaction with sev>msn was also examined, and it was found that all of them act as dominant suppressors of this genotype as well. It is interesting to mention that the Msn-induced defects in rhabdomere morphology are also suppressed by those deficiencies. These interactions suggest that the p38 kinases are redundant with JNK in the context of planar polarity signaling (Paricio, 1999).

Although genetic evidence suggests an involvement of bsk (JNK) and hep (JNKK) in polarity signaling, phenotypic analyses suggest that the JNK module components are highly redundant in this process. In the search for other kinases involved, it was found that deficiencies uncovering the genes encoding the recently described p38 MAP kinases (Dp38a and Dp38b), and the MAP kinase kinases (DMKK3 and DMKK4) dominantly suppress the sev>msn and sev-Dsh phenotypes, suggesting that these proteins also function downstream of Dsh and Msn in polarity signaling. It is interesting to note that all phenotypic defects of sev>Msn were dominantly suppressed by mutations in both components of the JNK and the p38 kinase module. In contrast to these interactions, tissue culture experiments in mammalian cells have shown that NIK overexpression leads to JNK phosphorylation, but no detectable p38 activation was observed. This difference can be explained by cell- and tissue-specific requirements, e.g. in Drosophila during dorsal closure, JNK activation downstream of Msn is not redundant, while redundancy and p38 interactions are observed in polarity signaling. Thus, it is tempting to speculate that both JNK and p38 kinases cooperate in polarity generation (Paricio, 1999).

The reported promiscuity of the kinases at both the MKK and the MAPK levels could account for the redundancy. The Drosophila MKKs and JNK/p38 MAPKs also appear to act (at least partially) on overlapping downstream targets. Whereas DMKK3 appears rather specific for p38 activation (although it activates both p38s), DMKK4 and Hep (the MKK7 cognate) both activate Bsk/JNK. Similarly, Bsk/JNK and both Dp38s can phosphorylate the downstream targets dJun and ATF2. Thus, a potential downstream target can still be phosphorylated when one of the upstream kinases is removed, and likewise for their upstream activators. An even more complicated picture may emerge when all relevant kinases are identified. Other examples of redundancy are described in yeast MAP kinases. Although KSS1 and FUS3 normally have specific roles in different pathways, it has been shown that they are redundant in the process of mating and in this case KSS1 replaces Fus3 when the latter is not present. The isolation and analysis of all the respective kinases and their mutants will be necessary to understand fully the contribution of each single kinase in planar polarity signaling (Paricio, 1999).

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date revised: 10 April 2009

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