Drosophila Development (PDF)

Model organisms for studying development traditional embryology focussed on egg-laying animals – development is ex utero, eggs are large, embryos can be experimentally manipulated (transplants, grafts etc) eg Xenopus laevis and the chicken contemporary research focuses on...

Model organisms for studying development traditional embryology focussed on egg-laying animals – development is ex utero, eggs are large, embryos can be experimentally manipulated (transplants, grafts etc) eg Xenopus laevis and the chicken contemporary research focuses on developmental genes – need animal suited for genetic analysis: large population size in lab, short generation time - the fruit-fly Drosophila melanogaster has provided most insight to date  Drosophila development: the body plan Genes that control development in Drosophila are very similar to those that control development in vertebrates. Drosophila is the best understood developmental system with great impact upon our knowledge of all development. (for example, Hox genes were first found in Drosophila.) Bilateral symmetry is established by the A/P and D/V axes. Early patterning occurs in the syncytial blastoderm and it becomes multicellular at the beginning of segmentation. In the syncytial blastoderm, concentration gradients of proteins (transcription factors) can diffuse, enter nuclei and provide positional information.  Drosophila development: maternal genes Maternal genes establish the body axes. Maternal gene products, mRNAs and proteins are expressed in the ovary. Zygotic genes are expressed by the embryo. About fifty maternal genes set up the A/P and D/V axes: the framework of positional information (spatial distributions of RNA and proteins). Zygotic genes respond to maternal gene expression. First broad regions are established, then smaller domains (with a unique set of zygotic gene activities) in a hierarchy of gene activity.  Drosophila development: the A/P axis Three classes of maternal genes set up the A/P axis Maternally expressed genes distinguish the anterior from the posterior. Maternal effect mutants result in females that can not produce normal progeny. Three mutant classes are 1) anterior, 2) posterior and 3) terminal classes. Anterior class: loss of head and thorax (sometimes replaced with posterior). Posterior class: loss of abdominal segments. Terminal class: missing acron and telson. bicoid, hunchback, oskar, nanos and caudal are key to A/P axis formation.  Drosophila development: maternal genes bicoid mRNA is sequestered in the oocyte during oogenesis. bicoid mRNA is localized to the anterior end of the unfertilized egg. After fertilization, the mRNA is translated and a concentration gradient is formed along the A/P axis. bicoid sets up a A/P morphogenic gradient and controls the first steps in embryo development and, thus, is essential to the developing organism.  Drosophila development: clues to the role of bicoid 1) bicoid (bcd) mutant females lay eggs that give rise to embryos missing the head and thorax. 2) Embryos missing anterior cytoplasm resemble above. 3) bcd embryos rescued by anterior cytoplasm injections. 4) Anterior cytoplasm can induce ectopic head & thoracic segments by injection in the middle of a bicoid egg. 5) in situ hybridization shows bcd mRNA is at the anterior part of the unfertilized egg (attached to cytoskeleton). 6) After fertilization, bcd mRNA is translated and protein forms A/P gradient. 7) bicoid: transcription factor and morphogen. 8) other anterior-group (group 1) maternal genes are involved in bicoid localization and translational control. Methods to determine localisation of specific mRNA and protein mRNA: in situ hybridization Chick embryo: expression of Pax6 localised to developing nervous system Methods to determine localisation of specific mRNA and protein protein: antibody staining permeabilize cells incubate with specific antibody conjugate visualize antibody binding 2 -maternal nanos blocks translation of maternal hunchback mRNA 2  A/P axis is divided into broad regions by gap genes The gap gene products, the first genes expressed along the A/P axis, are transcription factors (TFs). Gap gene expression is controlled initially by bicoid (=TF) in the synctial blastoderm. hunchback acts to help switch on the other gap genes (giant, Krüppel and knirps). Mutants of gap genes have large sections of the body pattern missing. Gap gene proteins are short lived (half-life of minutes) and extend only slightly outside of where the gene is expressed (bell-shaped concentration distribution.)  bicoid protein signals anterior hunchback expression Zygotic hunchback expression is in the anterior half of the embryo. Suppression in the posterior half produces a gradient running A to P. Anterior expression is switched on by high levels of bicoid. Increased anterior bicoid expression will result in extending the hunchback gradient toward the posterior half of the embryo. bicoid (homeodomain transcription factor) directly binds the hunchback promoter in several places. Expression of zygotic hunchback is activated by bicoid Method: Use reporter gene hooked up with developmental gene promoter Insert genetic construct into Drosophila embryo 2  Investigating interactions between transcription factors and promoters 1. Fuse part or all of promoter to a ‘reporter gene’, eg lacZ, GFP promoter gene promoter reporter gene 2. Create transgenic animal containing promoter- reporter gene fusion 3. Monitor expression of reporter gene product, eg histological stain for β-galactosidase; fluorescence microscopy for GFP 2  hunchback activates and represses other gap genes Krüppel is activated by a combination of bicoid and low levels of hunchback but is repressed by high levels of hunchback. This locates Krüppel expression to the centre of the embryo. knirps is repressed by high levels of hunchback. In this way the initial gradients of morphogens can lead to the establishment of regions within the syncytial blastoderm which themselves lead to the beginning of segmentation. 2