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Selva, E.M., and B.E. Stronach (2007) Germline clone analysis for maternally acting Drosophila hedgehog components. Methods Mol. Biol. 397:129-144 Many of the genes of Drosophila melanogaster have their transcripts deposited in developing oocytes. These maternally loaded gene products enable an otherwise homo-zygous mutant embryo to survive beyond the first stage of development for which the gene product is required. Zygotic mutations that disrupt the Hedgehog signal transduction pathway typically yield a segment polarity 'lawn of denticles' cuticle phenotype. However, an embryo homozygous mutant for a gene can achieve normal embryonic segmentation precluding classification of the gene as a component of the Hh pathway, if wild-type transcripts from the mother are present. This chapter discusses the theory and importance of analyzing germline clone embryos for maternally acting genes involved in Hh signal transduction, and describes in detail the method to generate mutant germline clone embryos. Polaski, S., L. Whitney, B.W. Barker, and B. Stronach (2006) Genetic analysis of Slipper/Mixed lineage kinase reveals requirements in multiple JNK-dependent morphogenetic events during Drosophila development. Genetics 174:719-733 Mixed Lineage Kinases (MLKs) function as Jun-N-terminal kinase (JNK) kinase kinases to transduce extracellular signals during development and homeostasis in adults. slipper (slpr), which encodes the Drosophila homolog of mammalian MLKs, has previously been implicated in activation of the JNK pathway during embryonic dorsal epidermal closure. To further define the specific functions of SLPR, we analyzed the phenotypic consequences of slpr loss- and gain-of-function throughout development, using a semi-viable maternal effect allele and wildtype or dominant negative transgenes. From these analyses we confirm that failure of dorsal closure is the null phenotype in slpr germline clones. In addition, there is a functional maternal contribution, which can suffice for embryogenesis in the zygotic null mutant, but rarely suffices for pupal metamorphosis, revealing later functions for slpr as the maternal contribution is depleted. Zygotic null mutants that eclose as adults display an array of morphological defects, many of which are shared by hep mutant animals, deficient in the JNK kinase (JNKK/MKK7) substrate for SLPR, suggesting that the defects observed in slpr mutants primarily reflect loss of hep-dependent JNK activation. Consistent with this, the maternal slpr contribution is sensitive to the dosage of positive and negative JNK pathway regulators, which attenuate or potentiate SLPR-dependent signaling in development. Though SLPR and TAK1, another JNKKK family member, are differentially used in dorsal closure and TNF/Eiger-stimulated apoptosis, respectively, a Tak1 mutant shows dominant genetic interactions with slpr suggesting potential redundant or combinatorial functions. Finally, we demonstrate that SLPR overexpression can induce ectopic JNK signaling and that the SLPR protein is enriched at the epithelial cell cortex.
Stronach, B. (2005) Dissecting JNK signaling, one KKKinase at a time. Dev. Dynam. 232:575-584 Specificity in signal transduction is essential to ensure distinct and appropriate cellular responses to extracellular cues. Determining the mechanisms that mediate specificity is key to understanding complex cell behaviors in development, when multiple pathways fire simultaneously and individual pathways are used recurrently. Jun kinase (JNK) signal transduction exemplifies a pathway that is used multiple times in animal development and homeostasis. Indeed, molecular genetic analysis of JNK signaling in Drosophila has shown that a core signaling module consisting of Hep (JNKK), Bsk (JNK), and Jun regulates various processes, including tissue morphogenesis, wound repair, stress response, innate immune response, and others. Six putative JNKK kinase (JNKKK) family members are present in the fly genome, which could activate the core module in response to distinct stimuli. The diversity of kinases at this level of the signaling hierarchy could substantially increase the number of possible signals that feed into activation of the core module. Recent studies have described the distinct phenotypic consequences of mutations in three of the genes, Slpr (dMLK), Tak1, and Mekk1. These data, together with Drosophila cell culture and genomic array analyses support the contention that the choice of JNKKK may contribute to signaling specificity in vivo. Whether this is achieved by individual JNKKKs or by means of a combinatorial mechanism will require a systematic characterization of compound mutants and a toolbox of transcriptional reporters specific for distinct JNK-dependent processes.
Stronach, B., and N. Perrimon (2003) JNK pathway in Drosophila morphogenesis. Pp 783-787 in Handbook of Cell Signaling, Bradshaw, R.A., and E. Dennis, Ed. Acaademic Press, San Diego, CA Renfranz, P.J., S.E. Siegrist, B.E. Stronach, T. Macalma, and M.C. Beckerle (2003) Molecular and phylogenetic characterization of Zyx102, a Drosophila orthologue of the zyxin family that interacts with Drosophila Enabled. Gene 305:13-26 Adherens junctions, which are cadherin-mediated junctions between cells, and focal adhesions, which are integrin-mediated junctions between cells and the extracellular matrix, are protein complexes that link the actin cytoskeleton to the plasma membrane and, in turn, to the extracellular environment. Zyxin is a LIM domain protein that is found in vertebrate adherens junctions and focal adhesions. Zyxin's molecular architecture and binding partner repertoire suggest roles in actin assembly and dynamics, cell motility, and nuclear-cytoplasmic communication. In order to study the function of zyxin in development, we have identified a zyxin orthologue in Drosophila melanogaster that we have termed Zyx102. Like its vertebrate counterparts, Zyx102 displays three carboxy-terminal LIM domains, a potential nuclear export signal, and three proline-rich motifs, one of which matches the consensus for mediating an interaction with Ena/VASP (Drosophila Enabled/Vasodilator-stimulated phosphoprotein) proteins. Here we show that Zyx102 and Enabled (Ena), the Drosophila member of the Ena/VASP family, can interact specifically in vitro and that this interaction does not occur when a particular mutant form of Ena, encoded by the lethal ena210 allele, is used. Lastly, we show that the zyx102 gene and Drosophila Ena are co-expressed during oogenesis and early embryogenesis, indicating that the two proteins may be able to interact during the development of the Drosophila egg chamber and early embryo.
Tan, C., B. Stronach, and N. Perrimon (2003) Roles of myosin phosphatase during Drosophila development. Development 130:671-681 Myosins are a superfamily of actin-dependent molecular motor proteins, among which the bipolar filament forming myosins II have been the most studied. The activity of smooth muscle/non-muscle myosin II is regulated by phosphorylation of the regulatory light chains, that in turn is modulated by the antagonistic activity of myosin light chain kinase and myosin light chain phosphatase. The phosphatase activity is mainly regulated through phosphorylation of its myosin binding subunit MYPT. To identify the function of these phosphorylation events, we have molecularly characterized the Drosophila homologue of MYPT, and analyzed its mutant phenotypes. We find that Drosophila MYPT is required for cell sheet movement during dorsal closure, morphogenesis of the eye, and ring canal growth during oogenesis. Our results indicate that the regulation of the phosphorylation of myosin regulatory light chains, or dynamic activation and inactivation of myosin II, is essential for its various functions during many developmental processes.
Stronach, B., and N. Perrimon (2002) Activation of the JNK pathway during dorsal closure in Drosophila requires the mixed lineage kinase, slipper. Genes Dev. 16:377-387 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.
Nie, W., B. Stronach, G. Panganiban, T. Shippy, S. Brown, and R. Denell (2001) Molecular characterization of Tclabial and the 3' end of the Tribolium homeotic complex. Dev. Genes Evol. 211:244-251 The homeotic selector gene labial is located at the 3' end of the Antennapedia complex (ANTC) and is required for proper head development in Drosophila. We have cloned and characterized the Tribolium ortholog of labial, Tclabial (Tclab). Similar to Drosophila labial, Tclab contains a single large intron upstream of the homeobox. In contrast, Tclab lacks an intron within the homeobox. The Tribolium ortholog of chaoptic, Tcchaoptic, transcribed from the opposite strand, is located immediately downstream of the homeotic complex, and its 3'UTR overlaps that of Tclab by 50 nucleotides. We have also sequenced the 13.5 kb interval between Tclab and maxillopedia (the Tribolium ortholog of Drosophila proboscipedia). In contrast to Drosophila, there is not a cluster of cuticle genes in this region. Finally, we have examined the expression of Tclab transcripts in Tribolium embryos. As previously described for Drosophila and other insects, the expression of Tclab is specific to the intercalary segment.
Stronach, B.E., and N. Perrimon (2001) Investigation of leading edge formation at the interface of amnioserosa and dorsal ectoderm in the Drosophila embryo. Development 128:2905-2913 The leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundary between two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that block LE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remains unclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, we have altered the extent of amnioserosa and dorsal ectoderm genetically, and assayed LE cell fate. We did not observe an expansion of LE fate in dorsalized or ventralized mutants. Furthermore, we observed that the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together our data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. We propose that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells.
Hong, Y., B. Stronach, N. Perrimon, L.Y. Jan, and Y.N. Jan (2001) Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. Nature 414:634-638 Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.
Stronach, B.E., P.J. Renfranz, B. Lilly, and M.C. Beckerle (1999) Muscle LIM proteins are associated with muscle sarcomeres and require dMEF2 for their expression during Drosophila myogenesis. Mol. Biol. Cell 10:2329-2342 A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.
Stronach, B.E., and N. Perrimon (1999) Stress signaling in Drosophila. Oncogene 18:6172-6182 Cells commonly use multiprotein kinase cascades to signal information from the cell membrane to the nucleus. Several conserved signaling pathways related to the mitogen activated protein kinase (MAPK) pathway allow cells to respond to normal developmental signals as well as signals produced under stressful conditions. Genetic and molecular studies in Drosophila melanogaster over the last several years have related that components of stress signaling pathways, namely the Jun kinase (JNK) and p38 kinase signaling modules, are functionally conserved and participate in numerous processes during normal development. Specifically, the JNK pathway is required for morphogenetic movements in embryogenesis and generation of tissue polarity in the adult. The role of the p38 pathway in generation of axial polarity during oogenesis has been inferred from phenotypic analysis of mutations in the Drosophila homolog of DMKK3. In addition to their requirement for normal development, cell culture and genetic investigations point to a role for both the JNK and p38 pathways in regulation of the immune response in the fly. This review details the known components of stress signaling pathways in Drosophila and recent insights into how these pathways are used and regulated during development and homeostasis.
Schmeichel, K.L., B.E. Stronach, and M.C. Beckerle (1998) Purification and assay of zyxin. Methods Enzymol. 298:62-76 A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and alpha-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture. Stronach, B.E., S.E. Siegrist, and M.C. Beckerle (1996) Two muscle-specific LIM proteins in Drosophila. J. Cell. Biol. 134:1179-1195 The LIM domain defines a zinc-binding motif found in a growing number of eukaryotic proteins that regulate cell growth and differentiation during development. Members of the cysteine-rich protein (CRP) family of LIM proteins have been implicated in muscle differentiation in vertebrates. Here we report the identification and characterization of cDNA clones encoding two members of the CRP family in Drosophila, referred to as muscle LIM proteins (Mlp). Mlp60A encodes a protein with a single LIM domain linked to a glycine-rich region. Mlp84B encodes a protein with five tandem LIM-glycine modules. In the embryo, Mlp gene expression is spatially restricted to somatic, visceral, and pharyngeal muscles. Within the somatic musculature, Mlp84B transcripts are enriched at the terminal ends of muscle fibers, whereas Mlp60A transcripts are found throughout the muscle fibers. The distributions of the Mlp60A and Mlp84B proteins mirror their respective mRNA localizations, with Mlp84B enrichment occurring at sites of muscle attachment. Northern blot analysis revealed that Mlp gene expression is developmentally regulated, showing a biphasic pattern over the course of the Drosophila life cycle. Peaks of expression occur late in embryogenesis and during metamorphosis, when the musculature is differentiating. Drosophila Mlp60A and Mlp84B, like vertebrate members of the CRP family, have the ability to associate with the actin cytoskeleton when expressed in rat fibroblast cells. The temporal expression and spatial distribution of muscle LIM proteins in Drosophila are consistent with a role for Mlps in myogenesis, late in the differentiation pathway.
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