Animal Development Drosophila axis formation Part 1 AP

Animal Development Drosophila axis formation Part 1: A-P patterning petros. ligoxygakis@bioch. ox. ac. uk

In this lecture: è The origin of Anterior-Posterior Axis è Mutant screens to isolate segmentation genes è Genetic analysis of early acting determinants è Important roles of post-transcriptional regulation and m. RNA/protein localisation è Methods of dissecting enhancers è Dosage-dependent activation of zygotic genes è Hierarchical organisation of segmentation genes

Developmental biology: Drosophila segmentation and repeated units * egg: generate the system * larva: eat and grow * pupa: structures in larvae grow out to form adult fly: metamorphosis (Drosophila is a holometabolous insect) February 06 1

Segmentation pattern èObvious segmentation begins to develop by germ band extension stage. èThe embryonic segmentation pattern has direct analogs to the final segments of the adult. èSegmentation pattern can be thought of as classical segments or midsegment-tomidsegment intervals called parasegments. èSome early embryonic segments become incorporated into the complex structures of the head and mouth.

Developmental biology: Drosophila segmentation and repeated units * Drosophila embryo: larva February 06 3

The Bcd gradient m. RNA (in situ) Protein (Ab staining)

bicoid protein gradient – gradient is interpreted at least at four different levels (thresholds).

Developmental biology: Drosophila segmentation and repeated units Embryo stage: developmental patterning: most obvious characteristics in resulting larvae: segments Segments are partially coated with hairs: denticles How do you get from “empty bag” to organized (segmented) larva? What is required? What gene Activities? Where and When? Fate map February 06 4

Developmental biology: Drosophila segmentation and repeated units Loss of gene activity will tell us what it is required for: Mutagenize flies and screen for segmental phenotypes (Nüsslein-Volhard and Wieschaus): Idea is to find all possible genes that when ko’d could lead to a developmental anomaly… Approach: randomly mutagenize whole genome; Isolate single gene mutants; Study their phenotypes (in embryo) 40. 000 individual mutations created: 150 very clear segmentation phenotypes…. February 06 5

Developmental biology: Drosophila segmentation and repeated units Segmental phenotypes (Nüsslein-Volhard and Wieschaus): * found 150 mutants that disrupt patterning in embryo on basis of cuticle defects. * amongst these, some have remarkable phenotypes: segments disappear in blocks gap mutants February 06 6

Developmental biology: Drosophila segmentation and repeated units February 06 6

Developmental biology: Drosophila segmentation and repeated units In gap mutants blocks of consecutive segments are deleted. gap gene B February 06 mutant phenotype caused by loss of gene activity Clone gap genes: determine expression domain Area of gene expression corresponds to affected body area in mutant 7

Developmental biology: Drosophila segmentation and repeated units * From gradients to large blocks of expression February 06 8

Developmental biology: Drosophila segmentation and repeated units gap genes expressed in broad regions. all encode transcription factors: targets? Answer comes from group of mutants found in same mutagenesis screen: February 06 9

Developmental biology: Drosophila segmentation and repeated units Knirps protein Fushi tarazu pair rule protein Only three blocks knirps action of gap gene expression: induces expression how to get of seven one ftz stripes? February 06 10

Developmental biology: Drosophila segmentation and repeated units And we have functional test: in knirps mutant, stripe should disappear…. in fact kruppel mutant February 06 11

Developmental biology: Drosophila segmentation and repeated units Gap transcription factor proteins, each induce one stripe Combined action of two gap proteins (or gap + head/tail proteins) define stripe each again. 3 To 7 Stripes! February 06 Hunchback protein Kruppel protein 12

Enhancer trapping in Drosophila P element recognition sites • Use transposon P element • Carries reporter gene e. g. TA b-galactosidase • Hops into genome P element vector • When lands near enhancer, activates gene expression • Expression similar to that of neighboring gene enhancer gene Y b-galactosidase gene Y

Regulation of eve stripes 3 and 7 February 06 eve 3 to 7 eve 3 and 7 hb-Z kni

The even-skipped (eve) stripes: a model to study how transcriptional regulation establishes positional information • Regulatory elements are fused to a lac. Z reporter gene and their expression is followed in transgenic flies. Eve has a long promoter element, which can be divided into regions that drive expression of various lac. Z-transgenes (and indeed expression of eve) in different stripes (stripe-elements). February 06

A great model for in vivo study of Transcription Factors and Their Binding Sites: The eve Stripe 2 Module Determine binding sites by footprinting Note that these transcription factors are the products of patterning genes higher in the regulatory hierarchy to eve. Determine importance of enhancer elements by mutagenesis.

Developmental biology: Drosophila segmentation and repeated units Trancript is made in nucleus: translated into cytoplasm. Transcription factor proteins are not transported out of cell: influence transcription in own nucleus February 06 In fly early embryo: transcription factor proteins can influence next door nuclei because there are no cell membranes to stop the proteins from diffusing. In fact, the diffusion is important, creates a gradient of activity: fine patterning. 13

Regulatory Protein Gradients That Control the Eve Stripe 2 Module The sharp Hunchback (green) and Kruppel (red) expression boundary.

Interaction Between Gene Products Sharpens Expression Boundaries early Sharpening eveftz stripes. late Autoactivation and mutual inhibition of the eve and ftz transcription factors Ftz Eve

Developmental biology: Drosophila segmentation and repeated units Transcription regulation of typical pair rule gene: highaffinity low February 06 coding region of pair rule gene 14

Developmental biology: Drosophila segmentation and repeated units pair rule genes = pair rule mutants! * in mutant embryo, every other segment is deleted. * pair rule gene expressed in seven stripes. In mutant: expression domains are deleted. * pair rule proteins are transcription factors (again) February 06 16

Developmental biology: Drosophila segmentation and repeated units Targets of pair rule transcription factor proteins? Pair rule protein wildtype wingless expression paired mutant February 06 segment polarity gene 17

Developmental biology: Drosophila segmentation and repeated units How: pair rule stripes segment polarity stripes Each stripe of pair rule protein defines a stripe of segment polarity gene expression. Each segment polarity gene is thus induced by two different sets of pair rule proteins (7 + 7 makes 14). Fine tuning of stripe limits (anterior) by overlap of pair rule proteins 14 stripes of segment polarity gene expression: 14 segments! February 06 18

Developmental biology: Drosophila segmentation and repeated units So far, transcription factors were able to define areas surrounding the nuclei were gene is expressed: no cell membranes. Expression domains of proteins Expression domains of genes February 06 19

Developmental biology: Drosophila segmentation and repeated units February 06 20

Developmental biology: Drosophila segmentation and repeated units Summary of early zygotic development in Drosophila embryos: Bicoid protein hunchback RNA Simple axis derived maternal system, divides egg into discrete units (mutants display loss of exactly the units). Transcription factor gradients create sets of “seven” identical units. February 06 21

Developmental biology: Drosophila segmentation and repeated units The action of the pair rule genes generates segmental stripes of expression of each segment polarity gene. In fact, pair rule protein action defines segments! Up until and including the pair rule proteins: all act within non-cellular environment, all are transcription factors. Segment polarity proteins act in cellular environment (not all transcription factors, become expressed at time of cellularization). February 06 22

Developmental biology: Drosophila segmentation and repeated units Further patterning within segments: next lecture February 06 23

Developmental biology: segment polarity genes and cell signalling b i c o i d g a p g e n e s time * transcription factors expressed in large blocks * induce expression of downstream transcription factors in smaller blocks * generates segmental expression (14) of segment polarity genes. February 06 1

A Gene’s Sphere of Influence Extends Only To Where Its Product is Expressed Maternal Coordinate Genes bicoid (bcd) bicoid(cad) (bcd) caudal (cad) hunchback (hb) hunchback nanos (nos) (hb) nanos (nos) Gap. Genes Pair-Rule Genes Segment Polarity Genes En hunchback (hb) hunchback Krüppel (Kr) (hb) Krüppel (Kr) knirps (kni) knirps giant (gt)(kni) giant (gt) tailless (tll) even-skipped (eve) odd-skipped (odd) hairy (h) runt (run) (ftz) fushi-tarazu paired (prd) (ftz) paired (prd) engrailed (en) wingless (wg)

Summary è Initial asymmetry for AP axis set up during oogenesis è Pattern organised by maternal proteins soon after fertilization è Localised maternal proteins activate or repress zygotic genes è Transcriptional and post-transcriptional regulation è Gradients of transcription factors are refined by mutual activation and repression and cell signalling.