Research Article

Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118 (2) :401-15. Abstract

We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Perkins LA, Larsen I, Perrimon N. corkscrew encodes a putative protein tyrosine phosphatase that functions to transduce the terminal signal from the receptor tyrosine kinase torso. Cell. 1992;70 (2) :225-36. Abstract

We describe the characterization of the Drosophila gene, corkscrew (csw), which is maternally required for normal determination of cell fates at the termini of the embryo. Determination of terminal cell fates is mediated by a signal transduction pathway that involves a receptor tyrosine kinase, torso, a serine/threonine kinase, D-raf, and the transcription factors, tailless and huckebein. Double mutant and cellular analyses between csw, torso, D-raf, and tailless indicate that csw acts downstream of torso and in concert with D-raf to positively transduce the torso signal via tailless, to downstream terminal genes. The csw gene encodes a putative nonreceptor protein tyrosine phosphatase covalently linked to two N-terminal SH2 domains, which is similar to the mammalian PTP1C protein.

Rutledge BJ, Zhang K, Bier E, Jan YN, Perrimon N. The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis. Genes Dev. 1992;6 (8) :1503-17. Abstract

We describe the molecular characterization of the Drosophila gene spitz (spi), which encodes a putative 26-kD, EGF-like transmembrane protein that is structurally similar to TGF-alpha. Temporal and spatial expression patterns of spi transcripts indicate that spi is expressed throughout the embryo. Examination of mutant embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. We propose that spi may act as a ligand for cell-specific receptors, possibly rhomboid and/or the Drosophila EGF receptor homolog.

1992_Genes Dev_Rutledge.pdf
Wieschaus E, Perrimon N, Finkelstein R. orthodenticle activity is required for the development of medial structures in the larval and adult epidermis of Drosophila. Development. 1992;115 (3) :801-11. Abstract

Lethal alleles of orthodenticle (= otd) cause abnormalities in the embryonic head that reflect an early role in anterior pattern formation. In addition, otd activity is required for the development of the larval and adult epidermis. Clonal analysis of both viable and lethal alleles shows that the adult requirement for otd is restricted to medial regions of certain discs. When otd activity is reduced or removed, some medial precursor cells produce bristles and cuticle characteristic of more lateral structures. Similar medial defects are observed in the larval epidermis of embryos homozygous for lethal otd alleles. Antibodies to otd recognize a nuclear protein found at high levels in the medial region of the eye antennal discs, the leg discs, the genital discs and along the ventral midline of the ventral epidermis of the embryo. These results suggest that the otd gene product is required to specify medial cell fates in both the larval and adult epidermis.

Dang DT, Perrimon N. Use of a yeast site-specific recombinase to generate embryonic mosaics in Drosophila. Dev Genet. 1992;13 (5) :367-75. Abstract

An efficient method for generating embryonic mosaics using a yeast site-specific recombinase (FLP), under the control of a heat shock promoter, is described. FLP-recombinase can promote mitotic exchange between homologous chromosomes that contain FRT (FLP Recombination Target) sequences. To demonstrate the efficiency of FLP-recombinase to generate embryonic mosaics, clones of the recessive and cell autonomous mutation armadillo (arm), detected by their ability to differentiate ectopic denticles in the naked cuticle of each abdominal segment, have been induced. We have analyzed the parameters of FLP-recombinase induced embryonic mitotic recombination and have demonstrated that clones can be efficiently induced during the postblastoderm mitotic divisions. We discuss applications of this technique for the analyses of the roles of various mutations during embryonic patterning.

1992_Dev Gene_Dang.pdf
Siegfried E, Chou TB, Perrimon N. wingless signaling acts through zeste-white 3, the Drosophila homolog of glycogen synthase kinase-3, to regulate engrailed and establish cell fate. Cell. 1992;71 (7) :1167-79. Abstract

Intrasegmental patterning in the Drosophila embryo is regulated by cell-cell communication. One of the signaling pathways that operates to specify positional information throughout the segment is mediated by the wingless (wg) protein, which is the homolog of the proto-oncogene Wnt-1. The early role of wg is to stabilize engrailed (en) expression by initiating a phase of en autoregulation in the adjacent more posterior cells. Here, we report that the segment polarity gene zeste-white 3 (zw3; also known as shaggy) acts as a repressor of en autoregulation. Genetic epistasis experiments indicate that wg signaling operates by inactivating the zw3 repression of en autoactivation. In addition, we demonstrate that zw3 encodes the Drosophila homolog of mammalian glycogen synthase kinase-3.

Kassis JA, Noll E, VanSickle EP, Odenwald WF, Perrimon N. Altering the insertional specificity of a Drosophila transposable element. Proc Natl Acad Sci U S A. 1992;89 (5) :1919-23. Abstract

Vectors derived from the Drosophila P element transposon are widely used to make transgenic Drosophila. Insertion of most P-element-derived vectors is nonrandom, but they exhibit a broad specificity of target sites. During experiments to identify cis-acting regulatory elements of the Drosophila segmentation gene engrailed, we identified a fragment of engrailed DNA that, when included within a P-element vector, strikingly alters the specificity of target sites. P-element vectors that contain this fragment of engrailed regulatory DNA insert at a high frequency near genes expressed in stripes.

Eberl DF, Perkins LA, Engelstein M, Hilliker AJ, Perrimon N. Genetic and developmental analysis of polytene section 17 of the X chromosome of Drosophila melanogaster. Genetics. 1992;130 (3) :569-83. Abstract

Polytene section 17 of the X chromosome of Drosophila melanogaster, previously known to contain six putative lethal complementation groups important in oogenesis and embryogenesis, has here been further characterized genetically and developmentally. We constructed fcl+Y, a duplication of this region, which allowed us to conduct mutagenesis screens specific for the region and to perform complementation analyses (previously not possible). We recovered 67 new lethal mutations which defined 15 complementation groups within Df(1)N19 which deletes most of polytene section 17. The zygotic lethal phenotypes of these and preexisting mutations within polytene section 17 were examined, and their maternal requirements were analysed in homozygous germline clones using the dominant female sterile technique. We present evidence that an additional gene, which produces two developmentally regulated transcripts, is located in this region and is involved in embryogenesis, although no mutations in this gene were identified. In this interval of 37 to 43 polytene chromosome bands we have defined 17 genes, 12 (71%) of which are of significance to oogenesis or embryogenesis.

Chou TB, Perrimon N. Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics. 1992;131 (3) :643-53. Abstract

We describe an efficient method for generating female germline mosaics by inducing site-specific homologous mitotic recombination with a yeast recombinase (FLP) which is driven by a heat shock promoter. These germline mosaics are produced in flies heterozygous for the agametic, germline-dependent, dominant female sterile (DFS) mutation ovoD1, where only flies possessing germline clones are able to lay eggs. This method, the "FLP-DFS" technique, is very efficient because more than 90% of females with germline clones can be recovered. We show that this heat-inducible, site-specific mitotic recombination system does not affect viability and that the germline clones recovered are physiologically the same as those created by X-ray induced mitotic recombination. We describe the parameters of FLP-recombinase induced germline mitotic recombination and the use of the "FLP-DFS" technique to analyze the maternal effect of X-linked zygotic lethal mutations.

Zhang K, Smouse D, Perrimon N. The crooked neck gene of Drosophila contains a motif found in a family of yeast cell cycle genes. Genes Dev. 1991;5 (6) :1080-91. Abstract

The crooked neck (crn) gene of Drosophila encodes a protein of 702 amino acids and contains 16 tandemly arranged copies of a 34-amino-acid repeat that is similar to the tetratrico peptide repeat (TPR). Multiple copies of the TPR motif have also been found in a family of yeast genes, including several members that are necessary for cell division. TPR-containing proteins encoded by the yeast genes CDC16, CDC23, and nuc2+ are required for progression through the G2/M transition of the cell cycle. Loss of zygotic expression of crn causes defects in the proliferation of brain neuroblasts and results in the absence of identified neuronal lineages in the central and peripheral nervous systems. The sequence similarity and mutant phenotypes are consistent with a cell cycle requirement for the crn gene product.

1991_Genes Dev_Zhang.pdf
Perrimon N, Noll E, McCall K, Brand A. Generating lineage-specific markers to study Drosophila development. Dev Genet. 1991;12 (3) :238-52. Abstract

To generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of beta-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.

1991_Dev Gene_Perrimon.pdf
Smouse D, Perrimon N. Genetic dissection of a complex neurological mutant, polyhomeotic, in Drosophila. Dev Biol. 1990;139 (1) :169-85. Abstract

Null mutations at the polyhomeotic locus of Drosophila produce a complex phenotype during embryogenesis, which includes death of the ventral epidermis, misregulation of homeotic and segmentation gene expression, and global misrouting of CNS axons. It is shown here, through the use of mosaic analyses, double mutant combinations, and in vitro culture experiments, that all aspects of the phenotype with the exception of the axonal phenotype are cell autonomous. The changes in homeotic and segmentation gene expression in the CNS are not caused by death of the ventral epidermis, but are cell autonomous effects which most likely cause changes in neuronal cell identity. The axonal phenotype associated with ph mutations is also independent of epidermal cell death, but may be due to the nonautonomous effects of altered neuronal identities or to death or transformation of some as yet unidentified cell type. Despite the apparent autonomy of the ph mutation, mutant neurons can influence the development of adjacent wild-type neurons, presumably by depriving them of their normal fasciculation partners.

1990_Dev Bio_Smouse.pdf
Finkelstein R, Smouse D, Capaci TM, Spradling AC, Perrimon N. The orthodenticle gene encodes a novel homeo domain protein involved in the development of the Drosophila nervous system and ocellar visual structures. Genes Dev. 1990;4 (9) :1516-27. Abstract

The orthodenticle (otd) locus of Drosophila is required for embryonic development, and null mutations of otd cause defects in head development and segmental patterning. We show here that otd is necessary for the formation of the embryonic central nervous system (CNS). otd mutations result in the formation of an abnormal neuropil and in the disappearance of identified neurons associated with the midline of the CNS. In addition, otd is allelic to ocelliless (oc), a mutation that causes the deletion of the ocelli of the adult fly. We have identified a transcription unit corresponding to the otd locus and find that it is expressed early in a stripe near the anterior pole of the cellular blastoderm and later in the region of the CNS from which these neurons normally arise. The predicted otd protein contains a well-conserved homeo domain and is therefore likely to be a transcriptional regulator involved in specifying cell fate both in the embryonic CNS and in the ocelli.

1990_Genes Dev_Finkelstein.pdf
Finkelstein R, Perrimon N. The orthodenticle gene is regulated by bicoid and torso and specifies Drosophila head development. Nature. 1990;346 (6283) :485-8. Abstract

In the Drosophila embryo, cell fate along the anterior-posterior axis is determined by maternally expressed genes. The activity of the bicoid (bcd) gene is required for the development of larval head and thoracic structures, and that of maternal torso (tor) for the development of the unsegmented region of the head (acron). In contrast to the case of thoracic and abdominal segmentation, the hierarchy of zygotically expressed genes controlling head development has not been clearly defined. The bcd protein, which is expressed in a gradient, activates zygotic expression of the gap gene hunchback (hb), but hb alone is not sufficient to specify head development. Driever et al. proposed that at least one other bcd-activated gene controls the development of head regions anterior to the hb domain. We report here that the homeobox gene orthodenticle (otd), which is involved in head development, could be such a gene. We also show that otd expression responds to the activity of the maternal tor gene at the anterior pole of the embryo.

Siegfried E, Perkins LA, Capaci TM, Perrimon N. Putative protein kinase product of the Drosophila segment-polarity gene zeste-white3. Nature. 1990;345 (6278) :825-9. Abstract

The metameric pattern of the Drosophila embryo is regulated by a combination of maternal and zygotic genes. The segment-polarity class of genes are required for the correct patterning within each segmental unit. Mutations in any one of these genes results in deletions and duplications of parts of each segment. The segment-polarity genes act coordinately by means of local cellular interactions to assign and maintain an identity for each cell in the segment, and to establish segment boundaries. Here we describe the molecular characterization of a novel segment-polarity gene, zeste-white3 (zw3). Embryos derived from germ lines that are homozygous for zw3 mutations (zw3 embryos) have phenotypes similar to embryos that are mutant for the segment-polarity gene naked (nkd). These embryos lack most of the ventral denticles, which are differentiated structures derived from the most anterior region of each segment. We have isolated the zw3 gene and compared the structure of one maternal and one zygotic transcript encoded by the gene. The zw3 gene is unique among the segment-polarity genes so far characterized, in that it encodes proteins that have homology to serine-threonine protein kinases. This indicates that zw3 may play a part in a signal transduction pathway involved in the establishment of cell identity within each embryonic segment.

Zhang K, Chaillet RJ, Perkins LA, Halazonetis TD, Perrimon N. Drosophila homolog of the mammalian jun oncogene is expressed during embryonic development and activates transcription in mammalian cells. Proc Natl Acad Sci U S A. 1990;87 (16) :6281-5. Abstract

By means of low-stringency cross-species hybridization to Southern DNA blots, human c-jun sequences were used to identify a unique Drosophila melanogaster locus (Djun). The predicted DJun protein is highly homologous to members of the mammalian Jun family in both the DNA binding and leucine zipper regions. Djun was mapped by in situ hybridization to position 46E of the second chromosome. It encodes a 1.7-kilobase transcript constitutively expressed at all developmental stages. Functionally, Djun in cooperation with mouse c-fos can trans-activate activator protein 1 DNA binding site when introduced into mammalian cells. Taken together, these data suggest that Djun, much like its mammalian homolog, may activate transcription of genes involved in regulation of cell growth, differentiation, and development. Furthermore, the identification of Djun allows one to exploit the genetics of Drosophila to identify genes in signal transduction pathways involving Djun and thus c-jun.

Perkins LA, Doctor JS, Zhang K, Stinson L, Perrimon N, Craig EA. Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster. Mol Cell Biol. 1990;10 (6) :3232-8. Abstract

The Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.

1990_Mol Cell Bio_Perkins.pdf
Perrimon N, Smouse D, Miklos GGL. Developmental genetics of loci at the base of the X chromosome of Drosophila melanogaster. Genetics. 1989;121 (2) :313-31. Abstract

We have conducted a genetic and developmental analysis of the 26 contiguous genetic complementation groups within the 19D3-20F2 interval of the base of the X chromosome, a region of 34 polytene bands delimited by the maroon-like and suppressor of forked loci. Within this region there are four loci which cause visible phenotypes but which have little or no effect on zygotic viability (maroon-like, little fly, small optic lobes and sluggish). There are 22 loci which, when mutated, are zygotic lethals and three of these, legless/runt, folded gastrulation and 13E3, have severe effects on embryonic development. In addition, three visible phenotypes have been defined only by overlapping deficiencies (melanized-like, tumorous head, and varied outspread). We have analyzed the lethal phases and maternal requirement of 58 mutations at 22 of the zygotic lethal loci by means of germline clone analysis using the dominant female sterile technique. Additionally, all lethal complementation groups, as well as a specific subset of deficiencies, have been studied histologically for defects in the development of the central and peripheral embryonic nervous systems.

Ng S-C, Perkins LA, Conboy G, Perrimon N, Fishman MC. A Drosophila gene expressed in the embryonic CNS shares one conserved domain with the mammalian GAP-43. Development. 1989;105 (3) :629-38. Abstract

By cross hybridization with the mammalian growth-related protein, GAP-43, we have isolated several Drosophila cDNAs and genomic sequences. These sequences correspond to a single copy gene that encodes two developmentally regulated transcripts 2.4 and 2.0 kb in length. The predicted protein sequence from the cDNAs contains a stretch of 20 amino acids closely related to the mammalian GAP-43 protein. These residues are also highly conserved in a cDNA isolated from the nematode C. elegans. Prior to dorsal closure, expression of the Drosophila gene is observed in non-neuronal tissues, especially in the mesectoderm and presumptive epidermis, both in a metameric pattern. After dorsal closure, expression becomes restricted to sets of cells that are segmentally reiterated along the periphery of the nervous system. These cells appear to include at least one specific set of glia that may establish scaffolding for the development of the longitudinal neuropile.

Ambrosio L, Mahowald AP, Perrimon N. l(1)pole hole is required maternally for pattern formation in the terminal regions of the embryo. Development. 1989;106 (1) :145-58. Abstract

Maternal expression of the l(1)pole hole (l(1)ph) gene product is required for the development of the Drosophila embryo. When maternal l(1)ph+ activity is absent, alterations in the embryonic fate map occur as visualized by the expression of segmentation genes fushitarazu and engrailed. If both maternal and zygotic activity is absent, embryos degenerate around 7 h of development. If only maternal activity is missing, embryos complete embryogenesis and show deletions of both anterior and posterior structures. Anteriorly, structures originating from labral and acron head regions are missing. Posteriorly, abdominal segments A8, 9 and 10, the telson and the proctodeum are missing. Similar pattern deletions are observed in embryos derived from the terminal class of female sterile mutations. Thus, the maternal l(1)ph+ gene product is required for the establishment of cell identities at the anterior and posterior poles of the Drosophila embryo.