Drosophila

Fruit fly (Drosophila melanogaster)

General characteristics -

Eukaryote, multicellular, diploid (3 autosomal chromosome pairs and one sex pair), rapid generation time (2 weeks), 300 offspring per female.

Sex determination XX(female), XY(male), ratio of X chromosomes to autosomes determines sex - X/A ratio. X/O is male, XXY is female

Polytene chromosomes in salivary glands, replicated chromosomes without mitosis, banding patterns of condensed and uncondensed chromatin, can map genes to specific regions, can be used to clone.

Many defined structures - body segments well characterized throughout development. Nervous system, visual system, behavioral characteristics

Advantages/Disadvantages

Genetics - well mapped genome by crossover frequencies and physical map (chromosome banding). Transposon mutagenesis - P element insertions can be regulated, insertional mutagenesis that can be mapped. Chromosome walking and clipping. Drawback - inability to identify redundant functions, large introns.

Molecular biology - enhancer traps help identify expression of genes in null mutants when lacZ replaces gene. In situ hybridization or immunodetection - allows for detection of spatial gene expression. Tight cell adhesion and shell casing are ideal for in situ analysis. Some DNA injection and transplantation experiments possible.

Enhancer trap technique - P element (transposon) with b-galactosidase (lacZ) gene with weak promoter. Integrates into genome and is regulated by near by enhancers. Might disrupt a gene but could also insert without phenotype. Can examine spatial pattern and perform chromosomal walk to identify genes potentially expressed in the same pattern.

Biochemistry - difficult due to lack of large cell pool and complexity. Drosophila cell cultures have been developed.

Embryogenesis - Extensive analysis of maternal and zygotic gene functions in determining anterior/posterior and dorsal/ventral axes. Saturating screens for mutations that affect patterns of development - identify maternal genes and zygotic genes.

- developmental gene hierarchy

- maternal genes can regulate anterior/posterior and dorsal/ventral and terminal.

- zygotic genes can regulate anterior/posterior by subsets - gap genes, pairrule genes, segment polarity genes.

-homeotic genes can regulate segment specification

- limitations of genetic approach - genes might be redundant, or essential for growth (housekeeping) or pleiotropic affect many parameters.

Maternal genes -

Anterior

- Bicoid gene. Anterior determination dependent on bicoid gene. Important for head and thoracic structures. Expressed in syncitium (before nuclei have been enclosed in separate cells. Bicoid RNA located in posterior and protein diffuses toward posterior forming a gradient. Increased dosages can alter positioning of other expressed genes (fushi tarazu - pair rule gene)

- encodes a transcription factor. Contains a homeo domain (misleading term for DNA binding protein). Known to bind to hunchback gene promoter. Hunchback promoter contains three bicoid binding sites (100,200,300 bp) upstream from transcription start site. Activates RNA polII transcription in a concentration dependent manner.

Posterior

-Nanos gene. Important for posterior genes. Represses hunchback which serves as a repressor of abdomen formation (see fig. 9). Repression of hunchback occurs by inhibiting translation of hunchback mRNA. Nanos mRNA becomes localized during embryogenesis (not localized during oogenesis) probably by way of a receptor.

-Vasa gene. Perhaps a regulator of Nanos mRNA. Has homology to a translation initiation factor. Protein becomes localized in posterior.

-BicaudalD gene. Perhaps localizes posterior factors. Contains homology to myosin heavy chain so it might be associated with the cytoskeleton

Terminal

-Torso gene. Required for terminal structure (acron and telson. Encodes a receptor tyrosine kinase. Located throughout the embryo but function might be localized by the distribution of the activating signal (product of torsolike gene) which is produced by somatic cells (follicle cells that surround the oocyte). Activity (possibly through a phosphorylation cascade) activates tailless gene as suggested by epistasis - neomorphic torso gene results in activity throughout embryo (doesn't need the signal) producing expanded terminal sections but this can be rescued by mutations in tailless.

Zygotic genes -

- use maternal positional information to produce segmental divisions.

-Gap genes - mutations in these genes result in large gaps in the segmental structure of the embryo. Examples - hunchback, Kruppel, and knirps. Now better defined as those genes affected by maternal factors and then regulate the next level of segmentation. hunchback, Kruppel, tailless, and knirps all encode transcription factors with Zn finger domains. Transcripts of these genes are localized but protein products are likely to diffuse over a greater area as suggested by the phenotypes of mutants. Regions of expression might overlap between different Gap proteins. Promoters that bind these factors may have sites for multiple different Gap proteins to bind.

-Pair rule genes - loss of these genes affects alternating segments. Examples - loss of T1,T3,A2,A4,A6,A8 in even skipped, loss of T2, and odd numbered belts in fushi tarazu, loss of etc.

- two classes of pair rule genes based on regulated expression (examined promoter sequences), primary- regulated by gap genes and each stripe may be regulated by a different enhancer element. examples are even skipped, hairy, and runt. secondary- regulated by perhaps other (primary) pair rule genes and all stripes are regulated by a single enhancer element. Perhaps needed for refinement of segmentation. Example is fushi tarazu. Expression in both classes occurs in open blastoderm or syncitium so factors can diffuse

- many pair rule genes might be transcriptional regulators based on homeoboxes or loop-helix-loop structures commonly associated with such factors.

-Segment Polarity Genes - loss of genes results in alterations in patterns within segments (definition between anterior and posterior regions) resulting in inverted duplications of the remaining structures.

- engrailed defines the anterior borders of each parasegment. encodes a putative transcription factor based on the presence of a homeobox.

- wingless defines the posterior borders of each parasegment, encodes a nonnuclear protein with regions important for secretion. Another gene patch is located in membranes.

- cellularization is complete when then genes are expressed so communication must occur cell-cell. Expression persists throughout embryonic development.

Dorsal Ventral System -

-Dorsal determination - cell fate depends on the maternally-acting dorsal gene which provides a morphogen required in a concentration dependent manner.

- dorsal protein or RNA does not exist in a gradient (asymmetrical) but uptake of protein occurs asymmetrically. Enters the nuclei on ventral side but not on the dorsal side.

- Toll gene encodes a putative membrane bound receptor activated by a signal from follicle cells (somatic cells). Can examine role of somatic cells and germline cells by transplantation experiments - transfer germline cells to another embryo. Genes required in the somatic cells for generating the signal that activates Toll include nudel, pipe, and windbeutel. Toll allows dorsal to act on ventral side and not on dorsal side establishing asymmetry.

Segmentation is further refined by homeotic genes such as Antennapedia (Antp) and Ultrabithorax (Ubx). Segment identity is further distinguished by the adult structures that arise from segment specific imaginal discs.

Generation of Pattern in Adult Flies

- patterns in differentiation generated during embryogenesis are further refined in the development of adult structures. Different segments in the larva carry imaginal discs that later develop into the adult structures.

Imaginal Discs (Imago = adult fly) - epidermal cells that give rise to most adult structures (wings, legs, antenna, etc.) Development of discs into final structures might depend on ecdysone (steroid-like) morphogen that is produced at high levels during molting (transitions between developmental states)

Transplantation experiments - Discs are somewhat cell autonomous in that they can be transferred to other flies or sites and still produce the same structure with the same cell number. Discs interact in an orientation dependent manner with the other epidermal cells. Can also dissociate cells of an imaginal disc and the cells will re-aggregate correctly and form the appropriate structure. Dissociated cells of different discs can be mixed but they segregate unless discs are similar and then crude mosaic structures can form (some segregation occurs in mosaic structure).

Regeneration experiments - can amputate structures and replace with structures from other sites. Healing is associated with new cell accumulation only when the fusion occurs at different areas of a segment. Suggests positional information is present in segments.

- segment also contain positional information in a circular axis. Can rotate segments of a leg and it will induce new dorsal/ventral boundaries creating two new legs at the junction.

Clonal analysis of imaginal disc development - can mix imaginal disc cells from different genetic backgrounds (can trace cells by markers - multiple wing hair mwh) or use gynandromorphs (females that lose one X chromosome during development to give clones of male tissue) that carry a recessive marker on only one of the X chromosomes. Can also use mitotic recombination induced by x-irradiation. Clones obey boundaries (eg. anterior/posterior) that are established during the development of the structure. Structures are made up of compartments (supracellular units) that are polyclonal but obey boundaries.

- development of epidermis structures (cuticle, chaetae or wing veins) occurs late. Cell fate established during the last few cell divisions rather than early on.

- development of homozygous minute clones (small slow cell growth) is not altered by the presence of wild-type cells in imaginal disc. Both genetic backgrounds are represented equally.

- function of segmental genes such as Ubx are required throughout entire development as late mitotic recombinants (homozygous mutants) during development show phenotypes

Segment identity and Homeotic genes - homeo refers to maintenance and homeotic mutations are described as those that alter the normal pattern of development - classic example is antennapedia mutation that results in the development of a leg where an antenna should be found. Homeotic mutations often involve the conversion of a segment into another segment- example T3 to T2 causes a second set of wings to appear where haltere wings should be.

- Segment identity appears to be regulated by a transcriptional cascade. Transcription factors regulate the expression of other transcription factors that regulate the expression of other transcription factors, etc.

- Homeotic Genes are those in which homeotic mutations occur. Many of these genes are transcription factors containing conserved DNA binding regions. Therefore the term homeodomain was given to the DNA binding regions.

- Segment identity often requires different hierarchies of transcription factors. Some sets of transcription factors are located in complexes.

Bx-C (bithorax complex) - encodes Ubx (ultrabithorax) and other genes. Historical perspectives - initial mutations in this region displayed a linear order on the chromosome that correlated with order of segment development. See Lewis model. Proposed a organized set of genes that were activated in an order that correlates with segment position. A little premature but valid in the sense it predicts a progressive increase in number of genes expressed in segments. Actually only three genes but many different phenotype produced depending if the mutation regulated gene expression (cis-acting enhancers), protein function, etc. Ubx gene is extremely large ~100 kb (average genes 1-3 kb) but much of this gene is intron sequence (regulated splicing) which might be important for delayed expression (1 hr lag time). Expressed over long periods of development - important for maintaining patterns rather than setting patterns.

Antp (Antennapedia) -

AS-C (achaete-scute gene complex)

-regulates the development of pattern elements that appear late in development (e.g., sensilla and chaetae)

- loss of function result in loss of elements in allele specific patterns, gain of function result in extra elements. Complete loss is not lethal to cell clones.

- contains four genes in a 100 kb region. All have helix-loop-helix domains

Eye development in Drosophila

Good structure to study since it is nonessential, easy to examine and has similar developmental parameters as other structures. Eyes consist of 700-750 facets packed in a honeycomb pattern to produce compound eye typical of insects.

The ommatidium is a 22 cell structure with 8 photoreceptor cells and 14 accessory cells. Photoreceptor cells are designated as R1-R8. Peripheral photoreceptors R1-R6 send axons to the first optic ganglion. R7 and R8 send axons to second ganglion.

- the eight cells arise from a recruitment of cells rather than a single cell precursor (determined by mosaic analysis). R7 is one of the last cells to be established.

Genes required for ommatidium development -

- sevenless (sev) - mutations result in ommatidium without R7 cell. R7 precursor differentiates into a nonneuronal cone cell. Wild type gene is required in the R7 cell - cell autonomous. Encodes a transmembrane receptor with a tyrosine kinase domain. Presumably receives a signal for development. Found in other cells but ligand is limited temporally and spatially. Constitutively active form of sev gene product causes the four presumptive cone cells to differentiate into R7 photoreceptors.

- bride-of-sevenless (boss) - gene believed to produce ligand for sevenless receptor. boss is only needed in R8 cells which come in contact with recruited R7 cell. Expression of boss in all cells by a heatshock promoter results in the presumptive cone cells to differentiate into R7-like cells.

- seven-up gene or rough gene are required for developmental switch that makes cells unresponsive to sevenless mediated events. Loss of these genes allows additional cells to become R7

- son of sevenless (sos) gene - encodes a guanine nucleotide release factor. Thought to act upstream of ras1 but downstream of sevenless. Isolated as a dominant suppressor of sevenless hypomorph (loss of function allele) - can not suppress a sevenless null allele.

Sex Determination and Dosage Compensation

Sex is determined by the ratio of the X chromosome to the autosomes (X/A ratio). Males ratio is less than one and females is 1 or greater. How does the organism sense the ratio? Product of the Sxl (sex lethal gene) get titrated out in males but not females. Transcript is expressed in both sexes but spliced differently (an exon in the males has a stop codon). Sxl splices its own transcript and and the tra trascript. The tra product and the tra2 product help splice the dsx transcript to give a female specific protein rather than the default male protein. Splicing cascade.

Dosage compensation - Sxl also regulates the expression of male-specific lethal (msl) genes which help hypertranscribe the single X chromosome in males. msl-1 gene binds to X chromosome and so does the mle (another msl type gene). So Sxl turns off male dosage compensation genes and turns on female somatic sex determination