Activation of the JNK pathway during dorsal closure in Drosophila requires the mixed lineage kinase, slipper

Abstract

The Jun kinase (JNK) pathway has been characterized for its role in stimulating AP-1 activity and for modulating the balance between cell growth and death during development, inflammation, and cancer. Six families of mammalian kinases acting at the level of JNKKK have emerged as upstream regulators of JNK activity (MLK, LZK, TAK, ASK, MEKK, and TPL); however, the specificity underlying which kinase is utilized for transducing a distinct signal is poorly understood. In Drosophila, JNK signaling plays a central role in dorsal closure, controlling cell fate and cell sheet morphogenesis during embryogenesis. Notably, in the fly genome, there are single homologs of each of the mammalian JNKKK families. Here, we identify mutations in one of those, a mixed lineage kinase, named slipper (slpr), and show that it is required for JNK activation during dorsal closure. Furthermore, our results show that other putative JNKKKs cannot compensate for the loss of slpr function and, thus, may regulate other JNK or MAPK-dependent processes.

Figure 1

Phenotypic effects of slpr mutations. (A) Embryonic cuticle phenotype of a strong loss-of-function slpr allele, 921, shows a large anterior dorsal hole compared with wild type (WT). (B) In situ hybridization to reveal dpp transcripts in wild-type (slpr⁹²¹/+) or mutant (slpr⁹²¹/Y) embryos at stage 11, germ band extended, and stage 13, germ band retracted. Stages according to Campos-Ortega and Hartenstein (1997). dpp expression is observed in cells at the dorsal edge of the ectoderm (arrowhead) in wild-type but not in slpr mutant embryos at both stages. Other aspects of dpp expression are normal. (C) A collection of embryos from a slpr⁹²¹ mutant stock has been labeled with anti-Fasciclin III antibodies to reveal the shape of cells in the dorsal ectoderm. The bracket marks leading edge (LE) cells at the margin that have begun to stretch and elongate as dorsal closure proceeds in both wild-type (slpr⁹²¹/FM7,ftzLZ) and slpr⁹²¹ mutant embryos. However, late stage slpr⁹²¹/Y mutant embryos exhibit a terminal phenotype in which LE cells fail to remain elongated and have rounded up, concomitant with slackening of the dorsal ectodermal sheet.

Figure 2

Epistasis analysis of slpr with JNK pathway components. (A) Embryonic cuticle preparation of progeny derived from the cross indicated and either maintained at 25°C (− heat shock) or subjected to a 30-min regimen at 37°C (+ heat shock) several hours prior to the beginning of dorsal closure. Embryos receiving heat shock express an inducible, constitutive active form of the cJun transcription factor that partially rescues the severe dorsal open phenotype of slpr⁹²¹ mutant embryos. (B) Embryonic cuticle preparation of progeny derived from the cross indicated. Inheritance of mutant Puc phosphatase, pucE69, a negative regulator of the JNK pathway, dominantly suppresses the severe slpr⁹²¹ mutant phenotype.

Figure 3

Mapping and cloning of the slpr locus reveal that slpr encodes a mixed lineage kinase. (A) Meiotic recombination mapping with recessive markers placed slpr to the right of singed (sn) in the 7DE region of the X chromosome. slpr complements the deletions, Df(1)C128 and Df(1)HA11 (+), but fails to complement Df(1)hl-a and Df(1)GE202 (−). These complementation data further refine the position of the slpr gene to the region between 7D13 and 7E2 excluding 7D18–7D20, which is deleted by Df(1)HA11 but complemented by slpr. Next, recombination mapping with P-element transposon insertions in the region (*) define slpr position within 7D13–7D18 (black box). A total of 2/3319 recombinant males were obtained between slpr and EP1167, whereas 0/2315 recombinants were obtained between slpr and EP1143. These results suggest that slpr is closer to the EP1143 marker, and thus likely to be found to the left of Df(1)HA11. Several genes in the region (Tbh, fs(1)h, mys, smox, Traf2, nAcRe, and Trf2) were useful landmarks to correlate the genetic and physical map with the genome sequence. (B) Among predicted and known genes in the defined region, we found a candidate gene encoding a protein kinase, CG2272. A genomic fragment encompassing CG2272 and closely neighboring CG15339, derived from the P1 clone DS08402, was used for slpr mutant rescue experiments. (C) Presence of the genomic transgene is sufficient to rescue the embryonic dorsal open phenotype of slpr. To identify the slpr mutant chromosome among the experimental population of embryonic cuticles, the slpr chromosome was marked with another recessive cuticle marker, gt.Alone, gt/Y embryos show selective loss of ventral denticles; gt,slpr/Y recombinant embryos display both ventral defects and a large dorsal hole. In the presence of the genomic transgene, P{slpr}, the dorsal open phenotype is rescued, whereas the gt phenotype is unaffected. (D) An EST clone, GH26507, derived from the CG2272 locus is 5129 nucleotides, containing 8 exons (black boxes) and 7 introns (lines) relative to genomic DNA. Genbank accession no. AY045717. The cDNA encodes an ORF of 1148 amino acids (see comment in Materials and Methods) with homology to mixed lineage kinases, which display an N-terminal SH3 domain, a catalytic kinase domain, leucine zipper motifs (LZ), and a Cdc42/Rac-interacting binding motif (C). slpr alleles, 921 and 3P5, each have a mutation within the kinase domain as indicated.

Figure 4

Slpr is related to human mixed lineage kinases among a larger group of JNKKK proteins. (A) Protein sequence alignment of fly MLK (Slpr) with human family members MLK2 (MST) and MLK3 (SPRK) in the N-terminal region encompassing the conserved domains as indicated above the sequence, SH3 (black line), kinase (gray line), LZ (dotted line), CRIB (double line). Residues highlighted in gray mark the sites in which slpr mutations are located. (B) Phylogenetic relationship of Slpr compared with the putative JNKKK protein kinase group. Relationship is based on kinase sequence only, using the CLUSTAL method with PAM250 residue weight. Five of six families are represented in the fly genome as a single homolog compared with human sequences. The MLK family shares ∼60% amino acid identity with Slpr. The kinases with leucine zipper motifs in the LZK branch are ∼36% identical to Slpr. Less related kinases in the JNKKK group are TAKs (∼25% identity), ASKs (∼20% identity), and MEKKs (∼18% identity), which show the greatest sequence divergence.

Figure 5

JNK pathway components lie genetically downstream of dRac1 in the adult eye. Expression of wild-type dRac1 under the control of the glass promotor (GMR-dRac1) in the Drosophila eye causes a rough appearance (top, left). Crossing w;GMR-dRac1 males to females that are wild type (+), or mutant for msn, slpr, hep, bsk, or dTAK tests for epistatic interactions. Shown are eyes from female progeny with the indicated heterozygous mutation in addition to one copy of GMR-dRac1. Reducing the gene dosage of msn, slpr, hep, and bsk by one-half significantly rescues the rough eye phenotype in comparison with w/+GMR-dRrac1/+. A puc mutation enhances the phenotype. Notably, loss of dTAK function, another putative JNKKK, does not suppress the GMR-dRac1-induced rough eye. Crosses were performed at 29°C. A wild-type eye is shown for comparison (red box, bottom, right).

Figure 6

Loss-of-function slpr mutant clones in adult tissues have normal epithelial planar polarity. Adult wings of wild-type (oreR) flies show a uniform polarized orientation of hairs, pointing distally downward (A). slpr clones marked by yellow (y) are only visualized in bristles at the margin (arrowheads), yet large y,slpr clones show normal orientation of hairs neighboring the margin (A‘). This is in comparison with the disrupted orientation of hairs in the dsh¹ wing (A“). Planar polarity is also evident in hairs of the adult notum. Clones of slpr mutant tissue are demarcated by y and singed (sn) (B) or only y (B‘) bristles (arrowheads). Smaller hairs in the region of the clone show normal polarized orientation, pointing posteriorly downward. In contrast, the hairs of dsh¹ mutant nota are randomly oriented, indicating a defect in planar polarity (B“).

Figure 7

slpr, encoding Drosophila MLK, is the MKKK for the Jun kinase pathway during dorsal closure in the fly. A canonical signaling pathway is indicated at left. Examples of mammalian proteins participating at each step are indicated at right. In Drosophila, dorsal closure involves signaling through the JNK pathway to control gene expression and cell sheet morphogenesis. Although the initiating signal is not known, it is likely that an early event is activation of dRac1, which by analogy to signal transduction with mammalian MKKKKs, participates with Msn to activate Slpr, the JNKKK. Sequential phosphorylation events through the kinase module, Slpr, Hep, and Bsk, lead to activation of dJun, regulation of gene expression, and cell sheet movement.