Role of Cka in imaginal disc growth and differentiation.

Weng, Li, and Wei Du*.  Committee on Cancer Biology, Ben May Institute for Cancer Research and Center for Molecular Oncology, University of Chicago, Chicago, IL 60637.  *wdu@huggins.bsd.uchicago.edu

      The c-Jun N-terminal kinase (JNK) signaling transduction pathway was initially identified in mammalian cells as a mediator of the cellular response to environmental stress (Hibi, Lin et al., 1993;  Derijard, Hibi et al., 1994).  Genetic studies in Drosophila have revealed that the conserved JNK signaling pathway regulates dorsal closure during embryonic development (Glise, Bourbon et al., 1995;  Riesgo-Escovar, Jenni et al., 1996; Sluss, Han et al., 1996;  Zeitlinger, Kockel et al., 1997). Dorsal closure is a process that begins at stage 13 of Drosophila embryogenesis.  During dorsal closure, the lateral epidermal cells elongate and move dorsally to enclose the entire embryo.  Mutations that block JNK pathway lead to a failure to express dpp (reviewed by Noselli and Agnes, 1999), a TGF-b family member that mediates concerted cell elongation during DC (Glise and Noselli, 1997; Riesgo-Escovar and Hafen, 1997; Riesgo-Escovar and Hafen, 1997; Sluss and Davis, 1997; Zeitlinger, Kockel et al., 1997) and lead to a dorsal closure defect (reviewed by Knust, 1997) .

      In addition to its role in directing the embryonic dorsal closure, JNK pathway is also required later in development for imaginal disc morphogenesis.  Imaginal discs are specialized small epithelial cell sacs that initiate during embryogenesis.  They proliferate and grow extensively during larval stages and undergo profound morphological changes at pupal stages to form the external adult structures (Cohen, 1993) .  One of the changes during metamorphosis is the fusion of the two lateral wing discs, giving rise to the dorsal thorax structure of the adult (Fristrom and Fristrom, 1993) .  This process, so-called “thorax closure”, is similar to the “dorsal closure” at embryo stage, which involves epithelial cell spreading.  Loss of JNK pathway activity leads to severe defects in disc morphogenesis, including small and malformed imaginal discs at larval stage and the absence or aberrant fusion of the two lateral wing discs during pupal stage (Agnes, Suzanne et al., 1999;  Zeitlinger and Bohmann, 1999;  Martin-Blanco, Pastor-Pareja et al., 2000).  The regulatory molecules required for thorax closure are similar to the ones directing dorsal closure in the embryo, indicating that the JNK signaling pathway may be widely used for controlling tissue closures during animal development.  The small imaginal disc phenotype indicates a growth defect in these tissues (Agnes, Suzanne et al., 1999) .  However, the underlying mechanism is still unknown.

Recently, Drosophila cka (connector of kinase to AP-1) was reported to function in the DJNK pathway (Chen et al., 2002).  cka deficient embryos display the typical dorsal-open phenotype associated with JNK pathway mutations ( Chen et al., 2002).  Here, we present a brief description of the characterization of the imaginal disc phenotypes of cka mutants.  We show that the cka mutants exhibit a small imaginal disc phenotype and a defect in neuronal differentiation.  Our study suggested that in addition to regulating dorsal closure at embryonic stage, CKA may also plays an important role in controlling the growth and cell differentiation during imaginal disc development.

In order to detect the expression of CKA protein, peptide corresponding to the C-terminal sequence of CKA was used to produce polyclonal antibodies.  Western blot experiments using fly embryonic and larval extracts showed that CKA is expressed in all developmental stages. The presence of CKA protein in early embryos (0-2 hr) suggests that this protein is maternally expressed (Figure 1A)

Two independent P element insertion lines, referred to as cka¹⁴⁵¹ and cka²⁰³⁹, were obtained from the Drosophila stock center.  PCR amplification and sequencing analysis revealed that both alleles have a P element inserted in the 5’ untranslated region of the CKA gene, about 1 kb upstream of the ATG site.  The cka¹⁴⁵¹ insertion disrupts the expression of the CKA gene, as shown by the Western blot assays in Figure 1B.  The protein level is greatly reduced but not completely removed in the zygotically homozygous mutant flies (Figure 1B).  It is possible the cka mutants are not null alleles or the maternal protein persists till late developmental stages. 

Animals zygotically homozygous or trans-heterozygous for these two alleles survive to pupal stages.  The pupal lethality results from disruption of the cka locus by P element insertion, as precise excision of the P element completely reversed the lethality.  Furthermore, ubiquitous expression of a transgene containing the full-length cka cDNA driven by a tublin promoter fully rescued the lethality of the homozygous or transheterozygous cka mutant flies.  These results demonstrated that the lethality associated with these two mutant alleles is due to disruption of CKA gene expression by P element insertions. 

Imaginal discs were dissected from third instar cka^1451/2039 larvae to characterize the role of CKA in imaginal disc development.  Interestingly, cka^1451/2039 mutant eye discs display a variable reduction ofdisc size as compared to wild type, suggesting a growth defect in the mutant imaginal discs (Figure 2).  In some of the cka^1451/2039 mutant imaginal discs, the overall morphology is normal, and the pattern of BrdU incorporation appears normal (Figure 2B and H).  Malformed and misfolded discs were also observed in the mutant flies, with a higher frequency in smaller discs (Figure 2C and F).  Similar phenotypes have been observed with mutations in DJNK signaling pathways, indicating that CKA may also function through the JNK pathway to regulate imaginal disc morphogenesis and growth during larval development. 

      The Drosophila compound eye is composed of approximately 800 repeating units called ommatidia, which consist of eight photoreceptor cells (R cells) and 12 non-neuronal accessory cells.  To determine if the cka mutation affects ommatidial development, we examined the expression of the protein Elav, a marker for neuronal cell differentiation (Robinow and White, 1991).  During the third larval instar, neuronal differentiation initiates at the morphogenetic furrow (MF), which is marked by a depression in the apical surface of the disc epithelium.  In the wild type eye disc, the Elav staining is first visible in the R8 cells immediately posterior to the morphogenetic furrow and continues as other photoreceptors are recruited into the cluster, reflecting a stepwise maturation of the growing cluster (Figure 2E).  These clusters of photoreceptor cells are well organized in rows.  Such expression pattern was disrupted in the cka mutant eye discs.  Elav expression was observed in the photoreceptor cells at the posterior portion of the cka mutant eye disc (Figure 2G and F).  However, the photoreceptor clusters are disorganized throughout the disc.  Furthermore, the anterior most Elav positive clusters already contain multiple differentiated photoreceptor cells (Figure 2G), indicating a differentiation defect during eye development in the cka mutant flies. 

Figure 2.  Imaginal disc phenotypes of zygotic cka mutant larvae.  (A-C), BrdU incorporation assay of a wild-type (A) and two cka1451/2039 mutant (B and C) third instar eye imaginal discs.  Black arrow, morphogenetic furrow; Black arrowhead, second mitotic wave.  (D and H), BrdU incorporation of a wild type (D) and a cka1451/2039 mutant (H) third instar wing discs.  White arrowhead, zone of non-proliferating cells (ZNC).  (E-G), Anti-Elav staining of wild type (E) and cka1451/2039.  (F-G), third instar eye imaginal phenotypes.  Panel G is an enlarged view of panel F.  Black arrows point to the anterior most ommatidial pre-clusters expressing Elav.

      In conclusion, phenotypic characterization of the cka¹⁴⁵¹ and cka²⁰³⁹ alleles revealed a novel role of the cka gene in regulating imaginal disc development.  The small imaginal disc phenotype indicates that CKA may function to regulate organ growth.  Furthermore, we observed neuron differentiation defects in the cka mutant during eye development.  This phenotype could be due to delayed differentiation of early photoreceptor cells (such as R8), or it could be due to precocious differentiation of late photoreceptor cells (such as R1 and R6).  Further investigations are needed to elucidate the mechanism by which CKA regulates eye cell differentiation.

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