Developmental biology: Drosophila segmentation and repeated units

1. 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) 1

2. Developmental biology: Drosophila segmentation and repeated units * Drosophila embryo: larva 3

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

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…. 5

5. * amongst these, some have remarkable phenotypes: segments disappear in blocks gap mutants 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. 6

6. Developmental biology: Drosophila segmentation and repeated units 6

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

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

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

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

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

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

13. Regulation of eve stripes 3 and 7 eve 1 and 7 eve 1 to 7 kni hb-Z

14. The even-skipped (eve) stripes: a model to study how transcriptional regulation establishes positional information • Regulatory elements are fused to a lacZ 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 lacZ-transgenes (and indeed expression of eve) in different stripes (stripe-elements).

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

16. high affinity Developmental biology: Drosophila segmentation and repeated units Transcription regulation of typical pair rule gene: coding region of pair rule gene low affinity 14

17. 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) 16

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

19. Developmental biology: Drosophila segmentation and repeated units pair rule stripes How: 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! 18

20. Expression domains of proteins Expression domains of genes 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. 19

21. Developmental biology: Drosophila segmentation and repeated units 20

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

23. 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). 22

24. Developmental biology: Drosophila segmentation and repeated units Further patterning within segments: next lecture 23