Drosophila single-minded represses gene transcription by activating the expression of repressive factors.

The Drosophila single-minded gene controls CNS midline cell development by both activating midline gene expression and repressing lateral CNS gene expression in the midline cells. The mechanism by which Single-minded represses transcription was examined using the ventral nervous system defective gene as a target gene. Transgenic-lacZ analysis of constructs containing fragments of the ventral nervous system defective regulatory region identified sequences required for lateral CNS transcription and midline repression. Elimination of Single-minded:Tango binding sites within the ventral nervous system defective gene did not affect midline repression. Mutants of Single-minded that removed the DNA binding and transcriptional activation regions abolished ventral nervous system defective repression, as well as transcriptional activation of other genes. The replacement of the Single-minded transcriptional activation region with a heterologous VP16 transcriptional activation region restored the ability of Single-minded to both activate and repress transcription. These results indicate that Single-minded indirectly represses transcription by activating the expression of repressive factors. Single-minded provides a model system for how regulatory proteins that act only as transcriptional activators can control lineage-specific transcription in both positive and negative modes.

Functional interactions between Drosophila bHLH/PAS, Sox, and POU transcription factors regulate CNS midline expression of the slit gene.

During Drosophila embryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent on Single-minded (Sim), a basic-helix-loop-helix (bHLH)-PAS transcription factor. We show here that Fish-hook (Fish), a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression. single-minded, fish-hook, and drifter are all expressed in developing midline cells, and both loss- and gain-of-function assays revealed genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from the slit gene and regulate the activity of this enhancer in cultured Drosophila Schneider line 2 cells. Fish-hook directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Fish can form homodimers and also associates with other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes.

Remembrance of things PAS: regulation of development by bHLH-PAS proteins.

Strange fits of passion I have knownellipsis (W Wordsworth, 'Strange fits of passion'.) bHLH-PAS proteins are regulators of developmental and physiological events that are well conserved between vertebrates and invertebrates. Recent studies using mouse knockouts of bHLH-PAS genes have provided novel insight into the roles of hypoxia inducible factors in controlling oxygen-regulated development and homeostasis, and the role of Single-minded-1 in regulating development and transcription in the hypothalamus. The Drosophila spineless and vertebrate Aryl hydrocarbon receptor bHLH-PAS orthologs both function in chemosensory processes, but in fundamentally different ways. Spineless controls antennal, limb, and sensory cell development, whereas the Aryl hydrocarbon receptor regulates the response to toxin metabolism. Structural analyses of the PAS domain provide insight into how this interaction domain can act as ligand-binding environmental sensor and signal transducer.

The CNS midline cells and spitz class genes are required for proper patterning of Drosophila ventral neuroectoderm.

The Drosophila embryonic central nervous system (CNS) develops from sets of neuroblasts (NBs) which segregate from the ventral neuroectoderm during early embryogenesis. It is not well established how each individual NB in the neuroectoderm acquires its characteristic identity along the dorsal-ventral axis. Since it is known that CNS midline cells and spitz class genes (pointed, rhomboid, single-minded, spitz and Star) are required for the proper patterning of ventral CNS and epidermis originated from the ventral neuroectoderm, this study was carried out to determine the functional roles of the CNS midline cells and spitz class genes in the fate determination of ventral NBs and formation of mature neurons and their axon pathways. Several molecular markers for the identified NBs, neurons, and axon pathways were employed to examine marker gene expression profile, cell lineage and axon pathway formation in the spitz class mutants. This analysis showed that the CNS midline cells specified by single-minded gene as well as spitz class genes are required for identity determination of a subset of ventral NBs and for formation of mature neurons and their axon pathways. This study suggests that the CNS midline cells and spitz class genes are necessary for proper patterning of the ventral neuroectoderm along the dorsal-ventral axis.

The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila.

The Drosophila spineless (ss) gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH-PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. In this report, we present evidence that Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo). We show that the ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss(-) mutations. The results of yeast two-hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus. Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. This suggests that the interaction of Ss and Tgo does not require additional signals, unlike the ligand-dependent interaction of Ahr and Arnt. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved.

Drosophila center divider gene is expressed in CNS midline cells and encodes a developmentally regulated protein kinase orthologous to human TESK1.

The Drosophila center divider gene (cdi) was isolated in an enhancer trap screen undertaken to identify genes involved in embryonic central nervous system (CNS) midline cell development. Three independent lines with P-element insertions at 91F were analyzed that all showed prominent beta-galactosidase expression in the CNS midline precursor cells and other cell types. Null mutations were created by imprecise P-element excision and shown to be larval lethal, although no severe CNS defects were observed in mutant embryos. The DNA surrounding the sites of insertion was cloned and found to contain a transcription unit that was dynamically expressed in a pattern corresponding to the enhancer trap line beta-galactosidase expression. Sequencing of cDNA clones revealed that the cdi gene encodes a 1140-amino acid protein that is an ortholog of the mammalian testis-specific TESK1 protein kinase. This serine/threonine kinase is distinct from other protein kinases because of sequence differences in the residues conferring substrate specificity. The unique sequence is conserved in Cdi, suggesting that Cdi/TESK1 represents a novel class of signaling proteins.

Regulation of bHLH-PAS protein subcellular localization during Drosophila embryogenesis.

The Drosophila Single-minded and Tango basic-helix-loop-helix-PAS protein heterodimer controls transcription and embryonic development of the CNS midline cells, while the Trachealess and Tango heterodimer controls tracheal cell and salivary duct transcription and development. Expression of both single-minded and trachealess is highly restricted to their respective cell lineages, however tango is broadly expressed. The developmental control of subcellular localization of these proteins is investigated because of their similarity to the mammalian basic-helix-loop-helix-PAS Aromatic hydrocarbon receptor whose nuclear localization is dependent on ligand binding. Confocal imaging of Single-minded and Trachealess protein localization indicate that they accumulate in cell nuclei when initially synthesized in their respective cell lineages and remain nuclear throughout embryogenesis. Ectopic expression experiments show that Single-minded and Trachealess are localized to nuclei in cells throughout the ectoderm and mesoderm, indicating that nuclear accumulation is not regulated in a cell-specific fashion and unlikely to be ligand dependent. In contrast, nuclear localization of Tango is developmentally regulated; it is localized to the cytoplasm in most cells except the CNS midline, salivary duct, and tracheal cells where it accumulates in nuclei. Genetic and ectopic expression experiments indicate that Tango nuclear localization is dependent on the presence of a basic-helix-loop-helix-PAS protein such as Single-minded or Trachealess. Conversely, Drosophila cell culture experiments show that Single-minded and Trachealess nuclear localization is dependent on Tango since they are cytoplasmic in the absence of Tango. These results suggest a model in which Single-minded and Trachealess dimerize with Tango in the cytoplasm of the CNS midline cells and trachea, respectively, and the dimeric complex accumulates in nuclei in a ligand-independent mode and regulates lineage-specific transcription. The lineage-specific action of Single-minded and Trachealess derives from transcriptional activation of their genes in their respective lineages, not from extracellular signaling.

Midline Fasciclin: a Drosophila Fasciclin-I-related membrane protein localized to the CNS midline cells and trachea.

Drosophila Fasciclin I is the prototype of a family of vertebrate and invertebrate proteins that mediate cell adhesion and signaling. The midline fasciclin gene encodes a second Drosophila member of the Fasciclin I family. Midline Fasciclin largely consists of four 150 amino acid repeats characteristic of the Fasciclin I family of proteins. Immunostaining and biochemical analysis using Midline Fasciclin antibodies indicates that it is a membrane-associated protein, although the sequence does not reveal a transmembrane domain. The gene is expressed in a dynamic fashion during embryogenesis in the blastoderm, central nervous system midline cells, and trachea, suggesting it plays multiple developmental roles. Protein localization studies indicate that Midline Fasciclin is found within cell bodies of midline neurons and glia, and on midline axons. Initial cellular analysis of a midline fasciclin loss-of-function mutation reveals only weak defects in axonogenesis. However, embryos mutant for both midline fasciclin and the abelson nonreceptor tyrosine kinase, show more severe defects in axonogenesis that resemble fasciclin I abelson double mutant phenotypes.

The Drosophila melanogaster similar bHLH-PAS gene encodes a protein related to human hypoxia-inducible factor 1 alpha and Drosophila single-minded.

The Drosophila melanogaster (Dm) similar (sima) gene was isolated using a low-stringency hybridization screen employing a Dm single-minded gene basic helix-loop-helix (bHLH) DNA probe. sima is a member of the bHLH-PAS gene family and the conceptual protein shares a number of structural features, including a bHLH domain, PAS domain, and homopolymeric amino acid stretches. Sima is most closely related to the human hypoxia-inducible factor 1 alpha bHLH-PAS protein. In situ hybridization experiments reveal that sima is transcribed in most or all cells throughout embryogenesis. It has been cytologically mapped to position 99D on the third chromosome, and is not closely linked to other known bHLH-PAS genes.

The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections.

Motor axons make synaptic connections with specific muscles, and this specificity unfolds during development as motoneuron growth cones make specific pathway choices and ultimately recognize and synapse on their specific muscle targets. The Drosophila clueless mutation was identified previously in a genetic screen for mutations that disrupt motoneuron guidance and connectivity. We show here that clueless is allelic to abrupt. The abrupt gene is required for the embryonic formation of specific synaptic connections between a subset of motoneurons and a subset of muscles. Mutations in abrupt also reveal its role in establishing and maintaining muscle attachments, adult sensory cell formation, and morphogenesis of adult appendages. The abrupt gene encodes a zinc finger protein with a conserved BTB domain. Abrupt is expressed in muscle nuclei but not motoneurons, suggesting that abrupt controls the muscle expression of molecules required for correct motoneuron targeting, as well as molecules required for correct muscle attachments.

Control of CNS midline transcription by asymmetric E-box-like elements: similarity to xenobiotic responsive regulation.

Central nervous system midline cells constitute a discrete group of Drosophila embryonic cells with numerous functional and developmental roles. Corresponding to their separate identity, the midline cells display patterns of gene expression distinct from the lateral central nervous system. A conserved 5 base pair sequence (ACGTG) was identified in central nervous system midline transcriptional enhancers of three genes. Germ-line transformation experiments indicate that this motif forms the core of an element required for central nervous system midline transcription. The central nervous system midline element is related to the mammalian xenobiotic response element, which regulates transcription of genes that metabolize aromatic hydrocarbons. These data suggest a model whereby related basic-helix-loop-helix-PAS proteins interact with asymmetric E-box-like target sequences to control these disparate processes.

Genetic analysis of the Drosophila single-minded gene reveals a central nervous system influence on muscle development.

The Drosophila single-minded gene is expressed in the embryonic central nervous system midline cells and plays a critical role in central nervous system development. Additional expression of single-minded is found in a subset of ventral muscle precursor cells. Null mutations of single-minded result in an alteration of the ventral oblique muscles, such that muscle fibers form inside the embryo above the central nervous system. This defect is due to the mislocalization of a subset of mesodermal precursor cells. The muscle defect observed in single-minded null mutations is not due to the absence of single-minded expression in muscle precursor cells and likely results from an influence of the central nervous system on ventral muscle development.

Transcriptional activation domains of the single-minded bHLH protein are required for CNS midline cell development.

The single-minded gene functions as a master developmental regulator within the midline cell lineage of the embryonic central nervous system of Drosophila melanogaster. Genetic experiments suggest that Single-minded can function as a transcriptional activator. Regions of the Single-minded protein were fused to the DNA binding domain of the mammalian transcription factor Sp1 and shown to activate transcription from a reporter gene linked to Sp1 binding sites. Three independent activation domains were identified in the carboxy terminal region of Single-minded that include areas rich in serine, threonine, glutamine and proline residues. Germ line transformation experiments indicate that the carboxy terminal activation domains, the PAS dimerization domain, and the putative DNA binding basic domain of Single-minded are required for expression of CNS midline genes in vivo. These results define in vivo a functional activation domain within Single-minded and suggest a model in which Single-minded activates transcription through a direct interaction with promoter elements of CNS midline genes.

Influence of Drosophila ventral epidermal development by the CNS midline cells and spitz class genes.

The ventral epidermis of Drosophila melanogaster is derived from longitudinal rows of ectodermal precursor cells that divide and expand to form the ventral embryonic surface. The spitz class genes are required for the proper formation of the larval ventral cuticle. Using a group of enhancer trap lines that stain subsets of epidermal cells, it is shown here that spitz class gene function is necessary for ventral epidermal development and gene expression. Analysis of single-minded mutant embryos implies that ventral epidermal cell fate is influenced by the CNS midline cells.

CNS midline enhancers of the Drosophila slit and Toll genes.

The Drosophila CNS midline cells comprise a small, well-characterized group of neurons and glia in which the transcriptional control of CNS development can be studied. Using germ-line transformation of lacZ fusion constructs, we have dissected putative regulatory regions of the slit and Toll genes to identify CNS midline-restricted transcriptional enhancers. This analysis has uncovered DNA regions able to drive lacZ expression in most tissues in which embryonic slit and Toll are expressed, including three separable CNS midline-conferring regions: one in the Toll gene which is expressed early in all of the CNS midline precursors, and two in the slit gene which are expressed later in the midline glia (MG).

The development and function of the Drosophila CNS midline cells.

1. The midline cells of the Drosophila embryonic CNS comprise a discrete neuroanatomical structure consisting of a small subset of neurons and glia. 2. Developmental commitment of the CNS midline cells requires the action of dorsal/ventral patterning genes. 3. The single-minded gene encodes a basic-helix-loop-helix transcription factor and acts as a master regulator for the CNS midline lineage. 4. A number of different transcription factors and proteins involved in cell-cell interactions are necessary for the differentiation of midline neurons and glia. 5. CNS midline cells have important functions in the formation of the ventral epidermis and axon commissures.

Dorsal-ventral patterning in Drosophila: DNA binding of snail protein to the single-minded gene.

The Drosophila snail gene is required for proper mesodermal development. Genetic studies suggest that it functions by repressing adjacent ectodermal gene expression including that of the single-minded (sim) gene. The snail gene encodes a protein with a zinc-finger motif, and here we report that the snail gene product is a sequence-specific DNA binding protein. The snail protein recognizes a 14-base-pair consensus sequence that is found nine times in a 2.8-kilobase sim regulatory region. These results provide evidence for the direct control of sim transcription by snail.

The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development.

Development of the Drosophila CNS midline cells is dependent upon the function of the single-minded (sim) gene. Sequence analysis shows that sim is a member of the basic-helix-loop-helix class of transcription factors. Cell fate experiments establish that sim is required for early events in midline cell development, including a synchronized cell division, proper formation of nerve cell precursors, and positive auto-regulation of its midline expression. Induction of ectopic sim protein under the control of the hsp70 promoter shows that sim can direct cells of the lateral CNS to exhibit midline cell morphology and patterns of gene expression. We propose that sim functions as a master developmental regulator of the CNS midline lineage.

The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells.

The single-minded (sim) gene of Drosophila encodes a nuclear protein that plays a critical role in the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the embryonic CNS. Using distinct cell fate markers, we observe that in sim mutant embryos the midline cells fail to differentiate properly into their mature CNS cell types and do not take their appropriate positions within the developing CNS. We further present evidence that sim is required for midline expression of a group of genes including slit, Toll, rhomboid, engrailed, and a gene at 91F; that the sim mutant CNS defect may be largely due to loss of midline slit expression; and that the snail gene is required to repress sim and other midline genes in the presumptive mesoderm.