Developmental Mutants in Drosophila
The embryonic development of an organism is determined by genes that may be active only during specific phases. Analysis of developmental mutants of embryos of the fruit fly Drosophila melanogaster has provided insight into the genetic regulation of developmental processes. Early developmental phases in embryos of very different organisms are regulated by similar genes.

• The segmental organization of the fruit fly (Drosophila melanogaster)
  
  The development of a fruit fly from the fertilized egg cell to the segmented body of the adult organism takes nine days. The larvae hatch after one day and pass through defined stages of embryonic development. The embryo forms a cocoon at five days and, after metamorphosis, emerges as a 2 mm-long adult fly. The head of the adult has three segments (C1–3), the thorax three (T1–3), and the abdomen eight segments (A1–8). A fruit fly has altogether 14 parasegments (P1–14), each corresponding to the last half of one and the first half of the next segment. The segmental organization is discernible in the larva.
  
  
• Embryonic lethal mutations
  
  Embryonic lethal mutations can be identified by appropriate crosses. One-fourth of the progeny of heterozygous flies (A/a) for an embryonic mutant gene (a) are homozygotes (aa) for the mutant allele (1). If a mutation involves maternal genes only (maternal effect), progeny of female homozygotes (bb) are lethally affected (2). Maternal effect genes code for early gene products that determine the polarity of the embryo; see below (C).
  
  
• Examples of developmental mutants
  
  Many developmental mutations are known in Drosophila melanogaster. They can be classified into different hierarchical gene classes. The normal larva (wild type) consists of three head segments, three thorax segments, eight abdominal segments, and the tail end (1). A mutation for anterior maternal effect, bicoid, leads to the development of a larva without head or thorax (2). Amutation of a gene called nanos affects the posterior end of the early larva. Gap genes establish the basic pattern of segmental organization. Mutations of the gap genes lead to omissions (gaps) in the segmental construction of the larva. In the Krüppel mutant (3), all thoracic and the abdominal segements 1–5 aremissing; in the Knirps mutant (4), abdominal segments 1–6 are absent. The genes for pair-rule determine the orientation and developmental fate of the 14 parasegments. Some mutations affect every second segment. With even-skipped (5), all even-numbered parasegments are missing. Mutation of the gene fushi tarazu leads to fewer than normal segments being formed (fushi tarazu is Japanese for too few segments). Segment polarity genes determine the polarity of each segment (7). There are more than ten segment polarity genes. Homeotic selector genes (8) determine the ultimate fate of each segment. With the mutant antennapedia (Ant), the antenna normally attached immediately under the eye is replaced by a leg (homeotic leg). (Figures adapted fromWatson et al., 1992).
  
  
• References
  
  Gehring,W.J., et al.: Homeodomain-DNA recognition.
  Cell 78:211–223, 1994.
  Kenyon, C.: If birds can fly, why can’t we?
  Homeotic genes and evolution. Cell 78:175–
  180, 1994.
  Lawrence, P.A.: The Making of a Fly. The Genetics
  of Animal Design. Blackwell Scientific,
  Oxford, 1992.
  Nüsslein-Volhard, C., Frohnhöfer, H.G., Lehmann,
  R.: Determination of anterior-posterior
  polarity in Drosophila. Science
  238:1675–1681, 1987.
  Watson, J.D., et al.: Recombinant DNA. 2nd ed.
  W.H. Freeman, Scientific American Books,
  1992.
  

• Hierarchy of developmental genes
  
  Four independent systems control the embryonic development of Drosophila melanogaster. A gradient of maternally derived protein coded for by the bicoid gene (bcd) determines development in the anterior region (head); likewise for nanos in the posterior region. They activate the gap genes. The three most important gap genes, hunchback, Knirps, and Krüppel, code for transcription factors of the zinc finger type (p. 218). Hunchback is expressed from the anterior part to about the middle of the embryo; Krüppel in the region of thoracic segments 4–6 and the first six abdominal segments; Knirps further posteriorly (parasegments 10–14). The gap genes induce the pair rule genes. Segment polarity genes determine the correct orientation of the individual segments. After segmentation is complete, selector genes determine the further development and the ultimate fate of the segments. They consist of three large gene complexes, the antennapedia complex (ANT-C), the bithorax complex (BX-C), and the ultrabithorax complex (UBX). Mutations of these gene complexes lead to unusual structures.
  
  
• Bithorax mutation
  
  A mutant for the bithorax complex (BX-C) causes the development of an additional thoracic segment with completely developed wings. (Photograph from Lawrence, 1992, after E. B. Lewis.)
  
  
• Structure of the antennapedia gene with homeobox
  
  Antennapedia is a gene of the antennapediacomplex (ANT-C), which is expressed in parasegments 5 and 6. It contains a segment of highly conserved DNA sequences in exon 8, the homeobox. Its sequence is identical in a wide variety of organisms, from Drosophila to mammals. It codes for about 60 amino acids (homeodomain). The homeodomain contains four domains of a helical protein with DNA-binding properties and functions as a transcription factor (Gehring et al., 1990).
  
  
• Homeotic genes in Drosophila and Hox genes in mouse`-aw31
  
  Homeobox genes also occur in vertebrates. A series of genes with the same anterior to posterior orientation and corresponding to the ANT and BX complexes (ANT-C and BX-C) are found in the embryonic brain of the mouse (and in man). The temporal expression corresponds to this orientation.
  
  
• Homeobox genes (HOX genes)
  
  In humans and in mice there are four groups (clusters) of genes that correspond to the homeo genes of Drosophila (HOX 1–4 in humans, Hox 1–4 in the mouse). Since mutations of these genes in mice lead to characteristic disorders, it is assumed that they are of clinical significance in humans also. The posterior end corresponds to the 5! end and the anterior end to the 3! end of the coding DNA.
  
  
• References
  
  Amores, A., et al.: Zebrafish hox clusters and
  vertebrate genome evolution. Science
  282:1711–1714, 1998.
  Gehring,W.J., et al.: The structure of the homeodomain
  and its functional implications.
  Trends Genet. 6:323–329, 1990.
  Krumlauf, R.: Hox genes in vertebrate development.
  Cell 78:191–201, 1994.
  Lawrence, P.A.: The Making of a Fly. The Genetics
  of Animal Design. Blackwell Scientific,
  Oxford, 1992.
  Marx, J.: Homeobox genes go evolutionary.
  Science 255:399–401, 1992.
  Reddihough, G.: Homing in on the homeobox.
  Nature 357:643–644, 1992.
  Scott, M.P.: A rational nomenclature for vertebrate
  homeobox (HOX) genes. Nucleic Acids
  Res. 21:1687–1688, 1993.

• Zebrafish embryos
  
  The zebrafish embryo is optically clear and can easily be studied by dissecting microscopy at maximally 80!magnification. At 29 hours after fertilization (pharyngula period), the main parts of the brain, the forebrain, midbrain, and hindbrain, are clearly visible along with the neural tube, several somites, and the floor plate. At 48 hours (hatching period) pigmentation begins, and the fins, eyes, brain, heart, and other structures become visible. At five days (swimming larva), the outline of a fish begins to show.
  
  
• Induced mutagenesis
  
  Adult fish (1) are used in a crossing scheme involving the exposure ofmale fish to 3mMethylnitrosourea (ENU) in an aqueous solution for three one-hour periods within one week. Mutagenized males are crossed with wild-type females (2). The first generation of this cross (F1) is heterozygous for one mutagenized genome. The next generation (F2) is raised from sibling matings, resulting in the presence of a mutation m in 50% of the F2 fish. Random matings involving two heterozygous parents results in 25% homozygous mutant offspring, which are analyzed. A total of 4264 mutants were identified this way by Haffter et al., 1996.
  
  
• Skeletal phenotype of the fused somites (fss) mutation
  
  
  Of the 1163 mutants characterized by their phenotypic effects, two examples are given here and in D. The fss mutants show irregular somite boundaries 72 hours after fertilization. At the seven-somite stage the only abnormality is the absence of somite boundaries. At later stages malformations of the tail vertebral column are visible (2). In wild-type fish the vertebral centrum has one neural arch dorsally and one hemal arch ventrally. In the fss mutant the arches are irregular in shape and, since they grow ectopically, in their relation to each other (2).
  
  
• The midbrain mutation no isthmus
  
  This is one example of the more than 60 mutant genes shown to affect the central nervous system and spinal cord (Haffter et al., 1996; Brand et al., 1996). The no isthmus (noi) mutation affects the boundary between midbrain and hindbrain. Normal embryos at the 24–48 h pharyngula stage show a conspicuous constriction at this boundary, whereas mutant noi embryos do not. In addition, the cerebellum derived from the posterior part is absent. Brand et al. (1996) studied the expression of two genes in this region, engrailed (eng) and wingless (wnt1). Normal embryos (wt) at 28 h show strong expression of eng between midbrain and hindbrain, while mutant noi embryos do not. Eightsomite- stage embryos double stained for eng and krx20 RNA, a marker for rhombomeres 3 and 5 in this region, show eng expression and krox20 expression at the midbrain–hindbrain boundary, but no expression of eng in the noi mutant. Similarly, wn1 expression posterior to the tectum (tec, the border of the two brain regions, in the 20-somite-stage embryo is eliminated in the mutant. (Figures adapted from Haffter et al., van Eeden et al., and Brand et al., 1996)
  
  
• References
  
  Dodd, A. et al.: Zebrafish: bridging the gap between
  development and disease. Hum. Mol.
  Genet. 9:2443–2449, 2000.
  Brand M., et al.: Mutations in zebrafish genes affecting
  the formation of the boundary between
  midbrain and hindbrain. Development
  123:179–190, 1996.
  Haffter P., et al.: The identification of genes with
  unique and essential functions in the
  development of the zebrafish, Danio rerio.
  Development 123:1–36, 1996.
  van Eeden, F.J.M., et al.: Mutations affecting
  somite formation and patterning in the zebrafish,
  Danio rerio. Development 123:153–
  164, 1996.

• Caenorhabditis elegans
  
  Caenorhabditis elegans is a small (about 1mm long), transparent worm with a life cycle of about 3 days. The basic structure is a bilaterally symmetric elongated body of nerves, muscles, skin, and intestine. It exists as one of two sexes: hermaphrodite or male. Hermaphrodites produce eggs and sperm and can reproduce by selffertilization. The adult hermaphrodite worm has 959 somatic cell nuclei; the adult male worm has 1031. In addition, there are 1000 to 2000 germ cells. The complete nucleotide sequence of the C. elegans genome was reported in 1998. The 97Mb DNA contains 19099 predicted genes, 12000 without known function. Coding sequences make up about 27% of the DNA, introns account for 26%. About 32% of the coding sequences are similar to human and about 70% of known human proteins have homologies in C. elegans. The largest group of genes are transmembrane receptors (790), in particular chemoreceptors; zinc finger transcription factors (480); and proteins with protein-kinase domains. (Diagram from Wood, 1988, after Sulston and Horvitz, 1977.)
  
  
• Embryonic origin of individual cells
  
  The developmental pathway of each individual cell can be traced. The various tissues arise from six founder cells. Of the 959 adult cells, 302 are nerve cells. Except for cells of the intestine and germ line, differentiated tissues stem from different founder cells. Cells with similar function are not necessarily related,while cells with different functions may be of the same origin. Genetically established rules determine the fate of the two daughter cells at each cell division.
  
  
• Mutations in the developmental control genes
  
  Many developmental control genes have been identified from analysis of ethyl methanesulfonate- induced point mutations. Somemutants determine an incorrect cell type (e.g., Z instead of B); others divide too early or too late (division mutants) resulting, for example, in C twice, instead of B and C.
  
  
• Programmed cell death
  
  Programmed cell death (apoptosis) is a normal part of vertebrate and invertebrate development. During embryonic development of C. elegans, one in eight somatic cells regularly dies at a defined time and branching point (1). The photographs (2) show the death of a cell (cell p11.aap) over a time span of about 40 minutes (from Wood, 1988, after Sulston and Horvitz, 1977). Mutations that interfere with programmed cell death can lead to severe disturbances. The human BCL-2 gene corresponds to the ced-9 gene in C. elegans, a gene that prevents apoptosis. The human BCL-2 gene codes for an inner mitochondrial membrane protein that acts by inhibiting cell loss by apoptosis in pro-B lymphocytes. Disruption of this gene on human chromosome 18 causes follicular lymphoma, a B-cell tumor.
  
  
• References
  
  The C. elegans Sequencing Consortium: Genome
  sequence for the nematode C. elegans: A
  platform for investigating biology. Science
  282:2012–2018, 1998.
  Culetto, E., Sattelle, D.B.: A role for Caenorhabditis
  elegans in understanding the function
  and interactions of human disease genes.
  Hum. Mol. Genet. 9:869–878, 2000.
  W.B.Wood and the Community of C. elegans Researchers:
  The Nematode Caenorhabditis
  elegans. Monograph 17, Cold Spring Harbor,
  New York, 1998.

• Normal development and structure
  
  The basic structural plan can be understood as an axial and a radial pattern superimposed on each other. An octant stage, a globular stage, and a so-called heart stage can be differentiated before the seedling is formed. The regions A, C, and B of the octant stage correspond to the regions A, C, and B of the heart stage. Region A forms the cotyledon and the meristem; C forms, the hypocotyl region; and B forms the root. The seedling consists of a set of identifiable structures including vessels (v), external epidermis (e), short meristem (s), cotyledons (c), and hypocotyl (h), ground tissue (g), and root primordium (r) at the bottom (labeled v instead of r). In the heart stage, the essential organization of the plant is predetermined.
  
  
• Deletions in the apical–basal pattern
  
  The mutations can be induced by 0.3% ethyl methanesulfonate. Using complementation analysis, Mayer et al. (1991) determined mutations in three areas of the plant. These mutations affect the apical–basal pattern, the radial pattern, and the shape. Apical–basal deletions involve one of several genes, each leading to a characteristic phenotype: apical deletion (Gurke), central deletion (Fackel), basal deletion (monopteros), and terminal deletion (Gnom).
  
  
• Wild-type
  
  The normal structure of embryonic Arabidopsis results from two basic processes: formation of patterns (apical–basal and radial orientation) and morphogenesis through different cell forms and regional differences in cell division.
  
  
• Phenotypes of embryonic mutants
  
  The four mutant phenotypes in the apical–basal pattern are Gurke (9 alleles), Fackel (5 alleles), monopteros (11 alleles), and Gnom (15 alleles) (see B). Deletions in the radial pattern lead to phenotypes Keule (9 alleles) and Knolle (2 alleles). Mutants of shape are Fass (12 alleles), Knopf (6 alleles), and Mickey (8 alleles). (Photographs from Mayer et al., 1991.) The monopteros gene (ml) is apparently very important for apical— basal development. However, it also has an indirect effect on the spatial arrangement of the apical structures. It is not necessary for root development (Berleth and Jürgens, 1993).
  
  
• References
  
  Berleth, T., Jürgens, G.: The role of the monopteros
  gene in organising the basal body region
  of the Arabidopsis embryo. Development
  118:575–587, 1993.
  Jürgens, G.: Memorizing the floral ABC. Nature
  386:17, 1997.
  Lin, X., et al.: Sequence and analysis of chromosome
  2 of the plant Arabidopsis thaliana. Nature
  402:761–768, 1999.
  Mayer, U., et al.: Mutations affecting body organization
  in the Arabidopsis embryo. Nature
  353:402–407, 1991.
  Pelaz, S. , et al.: B and C floral organ identity
  functions require SEPALLATA MADS-box
  genes. Nature 405:200–203, 2000.
  Sommerville, C., Sommerville, S. : Plant
  functional genomics. Science 285:380–
  383, 1999.